CN112218951B - Wheat blue grain gene and application thereof - Google Patents

Abstract

A wheat blue grain gene and application thereof belong to the fields of plant molecular biology, biochemistry, genetics and plant breeding. By utilizing the differential expression analysis of blue-grain and white-grain wheat, four genes for controlling the blue-grain character of the wheat are obtained: two MYB family transcription factors and two bHLH family transcription factors, and provides a plant recombinant expression vector of the genes and a method for regulating and controlling the synthesis of plant anthocyanin, and the two bHLH family transcription factors have important theoretical and practical significance for researching the synthetic route of aleurone layer pigment of the blue-grain wheat, serving as a screening marker in the process of plant transformation and improving the nutritive value of plants.

Description

Wheat blue grain gene and application thereof

Technical Field

The invention relates to the field of plant molecular biology, biochemistry, genetics and plant breeding, in particular to a gene for regulating and controlling the blue grain character of wheat. The invention provides nucleic acid molecules of four wheat blue grain character related genes, a plant recombinant vector and an application method of the genes or the vector.

Technical Field

There are two colors naturally found in common wheat grains: white or red grains, and very few blue or purple grains. Blue-grain or purple-grain wheat can be used as an important genetic marker to be applied to genetic breeding of wheat, particularly, in China, the application research of blue-grain wheat is relatively deep, for example, a 4E-ms hybrid wheat production system successfully utilizes blue-grain characteristics to effectively maintain wheat nuclear male sterility (Zhou et al, 2006).

The outer surface of the endosperm of the wheat grain is wrapped with three layers of tissues from outside to inside, which are respectively as follows: the color of the wheat seed is determined by different anthocyanidin accumulated in different tissues. Purple-grain wheat is derived from purple anthocyanin in the outermost pericarp, and the pericarp is developed by a female parent, so that the purple-grain shape presents a maternal inheritance pattern. Blue-grain wheat is derived from blue anthocyanin of aleurone layer, mainly generated by distant hybridization between common wheat and other species, and the sources of the blue-grain wheat are two: spike length couchWheat straw (Thinopyrum ponticum) and wild wheat (Triticum monococcum). In the sixties to the eighties of the last century, many scholars at home and abroad, such as Knott, sharman, lizhen and the like, obtain blue grain wheat from filial generations of elytrigia elongata and common wheat, and prove that the 4E (also named 4 Ag) chromosome of the elytrigia elongata carries blue grain genes. Plum tremolo et al, through genetic analysis, believe that the blue grain gene is genetically stable and independent, with significant dose effects, with its blue grain trait closely resembling genetic control of a pair of genes (plum tremolo et al, 1982). GISH and FISH experimental analysis of a series of different blue-grain translocation lines further mapped the blue-grain genes within the 0.71-0.80 segment of the long arm of the 4Ag chromosome (distance from the centromere) (Zheng et al, 2006). In 1990, keppenne named Blue aleurone (Ba) gene from a Blue aleurone layer gene of elytrigia repens, and foreign scholars thought that the Blue trait is controlled by two complementary genes together. Joppa et al in 1982 demonstrated that the Blaukom series derived from Triticum aestivum is formed by replacement of the 4A or 4B chromosome of Triticum aestivum by a pair of 4A chromosomes of Triticum aestivum, 4A from Triticum aestivum ^m The chromosomes are non-homologous to the 4A chromosome in tetraploid and hexaploid wheat. Kuspiral et al named the blue aleurone layer gene from wild one-grain wheat as Ba2 gene in 1989, and the Ba2 gene was located at 4A ^m Near the centromere of the long arm of the chromosome (Dubcovsky et al, 1996).

Genes controlling red grain and purple grain traits in wheat have been cloned, and there is no report on blue grain traits. In 2005, himi et AL reported that the red-grain-like major genes R1, R2 and R3 located on the 3AL, 3BL and 3DL chromosomes encode 3 highly homologous Myb family transcription factors (Himi et AL, 2005). Genetic analysis of Triticum aestivum showed that purple-grain trait is controlled by a Pp-1 (purple pericarp) site located on the short arm of chromosome 7 and a Pp3 site located on chromosome 2AL, wherein Pp-1 encodes a Myb family transcription factor and Pp3 encodes a Myc family transcription factor containing a bHLH (basic helix-loop-helix) domain (Khlestkina et AL,2013, shoeva et AL, 2014.

Anthocyanidin for determining wheat grain color is a water-soluble secondary metabolite-flavonoid compound, and is widely distributed in roots, stems, leaves, flowers, fruits and seeds of higher plants. Plant anthocyanin synthesis belongs to a branch of flavonoid synthesis pathway, and main synthetases in the pathway comprise chalcone synthetase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR) and the like (Gongkuai et al, 2011), and all the genes are structural genes for anthocyanin synthesis. The regulation and control of anthocyanin synthesis are mainly completed by three types of transcription factors: MYB, bHLH and WD40. Most of the anthocyanidins in the species are directly regulated and activated by a protein complex formed by compounding the three transcription factors, and a few anthocyanidins can be synthesized by only a single regulation factor.

The blue-grain wheat is an ideal morphological characteristic for wheat marker character and wheat cytogenetics research, and is also an important basic material in wheat chromosome engineering research. The invention utilizes the differential expression analysis of blue grain and white grain wheat to obtain 4 genes for controlling the blue grain character of wheat: two MYB family transcription factors and two bHLH family transcription factors. The invention is beneficial to researching the synthesis way of the blue-grain wheat aleurone layer pigment, can be used as a screening marker in the plant transformation process, and can increase the synthesis of anthocyanin by expressing the gene in the plant, thereby improving the nutritive value of the plant.

Disclosure of Invention

All references mentioned herein are incorporated herein by reference.

Unless defined to the contrary, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless indicated to the contrary, the techniques used or referred to herein are standard techniques well known to those of ordinary skill in the art. The materials, methods, and examples are illustrative only and not intended to be limiting.

The invention provides a blue grain gene with an anthocyanin synthesis regulation function, wherein the names of the blue grain genes are ThMYB1, thMYB2, thR 1and ThR2 respectively, and the blue grain gene can change tissues or organs at the expression part of the blue grain gene into blue. Wherein the genome nucleotide sequence from the start codon to the stop codon of the ThMYB1 gene is shown as SEQ ID NO. 1, the nucleotide sequence of a coding region (CDS) of the ThMYB1 gene is shown as SEQ ID NO. 2, and the amino acid sequence coded by the CDS is shown as SEQ ID NO. 3. Wherein the genome nucleotide sequence from the start codon to the stop codon of the ThMYB2 gene is shown as SEQ ID NO. 4, the nucleotide sequence of a coding region (CDS) of the ThMYB2 gene is shown as SEQ ID NO.5, and the amino acid sequence coded by the CDS is shown as SEQ ID NO. 6. Wherein the genome nucleotide sequence from the start codon to the stop codon of the ThR1 gene is shown as SEQ ID NO. 7, the nucleotide sequence of the coding region (CDS) thereof is shown as SEQ ID NO. 8, and the amino acid sequence coded by the CDS thereof is shown as SEQ ID NO. 9. Wherein the genome nucleotide sequence from the start codon to the stop codon of the ThR2 gene is shown as SEQ ID NO. 10, the nucleotide sequence of the coding region (CDS) thereof is shown as SEQ ID NO. 11, and the amino acid sequence coded by the CDS thereof is shown as SEQ ID NO. 12.

As will be appreciated by those skilled in the art, the locus coeruleus genes described herein also include nucleotide or protein sequences that are highly homologous to the nucleotide or protein sequences of the ThMYB1, thMYB2, thR 1and ThR2 genes and that have the same function of regulating plant anthocyanin synthesis. The homologous gene which is highly homologous and has the anthocyanin synthesis regulation function comprises a DNA sequence which can be hybridized with DNA with the sequence shown in SEQ ID NO. 1, 2, 4, 5, 7, 8, 10 or 11 under the strict condition. Or nucleotide sequence with 85% similarity between the coded amino acid sequence and the protein amino acid sequence shown in SEQ ID NO. 3, 6, 9 or 12. As used herein, "stringent conditions" are well known and include, for example, hybridization in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA at a temperature of preferably 53 ℃ to 60 ℃ for a hybridization time of preferably 12 to 16 hours, followed by washing with a washing solution containing 0.5 XSSC, and 0.1% SDS at a washing temperature of preferably 62 ℃ to 68 ℃ for a washing time of 15 to 60 minutes.

The homologous gene also comprises a DNA sequence which has at least 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with the full length of the sequence shown in SEQ ID NO. 1, 2, 4, 5, 7, 8, 10 or 11 and has the function of regulating the synthesis of plant anthocyanin, and can be separated from any plant. The percentage of sequence similarity can be obtained, among others, by well-known bioinformatics algorithms, including the algorithms of Myers and Miller, needleman-Wunsch global alignment, smith-Waterman local alignment, pearson and Lipman similarity search, karlin and Altschul. As is well known to those skilled in the art.

The invention also provides an expression cassette, which contains the DNA sequence of the blue grain gene disclosed by the invention, and the nucleotide sequence of the blue grain gene is selected from one of the following sequences:

(a) As shown in SEQ ID NO: 1. 2, 4, 5, 7, 8, 10 or 11;

(b) The coded amino acid sequence is shown as SEQ ID NO: 3. 6, 9 or 12;

(c) A DNA sequence capable of hybridizing to the DNA of the sequence of (a) or (b) under stringent conditions; or

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences described in (a) to (c) and having anthocyanin synthesis-controlling function; or

(e) A DNA sequence complementary to any one of the sequences of (a) to (d).

Specifically, the blue-particle gene in the above expression cassette is also operably linked to a promoter capable of driving its expression, including but not limited to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a spatio-temporal specific expression promoter. The constitutive promoter has no tissue and time specificity in gene expression, and external factors hardly influence the exogenous gene expression started by the constitutive promoter. The constitutive promoter includes, but is not limited to, caMV35S, FMV35S, rice Actin (Actin 1) promoter, maize Ubiquitin (Ubiquitin) promoter, and the like. The tissue-specific promoter disclosed by the invention comprises due general promoter elements, and also has the characteristics of an enhancer and a silencer, and the promoter has the advantages that the expression of genes at a specific tissue part of a plant can be started, the unnecessary expression of exogenous genes is avoided, and the whole energy consumption of the plant is saved. The tissue-specific promoter includes, but is not limited to, LTP2 seed-specific expression promoter, END2 seed-specific expression promoter, aleurone layer-specific expression promoter, and the like. The inducible promoter of the present invention refers to a promoter that can greatly increase the transcription level of a gene under the stimulation of some specific physical or chemical signals, and the inducible promoters that have been isolated at present include, but are not limited to, stress inducible expression promoters, light inducible expression promoters, heat inducible expression promoters, wound inducible expression promoters, fungus inducible expression promoters, symbiotic bacteria inducible expression promoters, and the like.

The above expression cassette of the present invention may further comprise a screening gene, and the screening gene may be used to screen out plants, plant tissue cells or vectors containing the expression cassette. The screening gene includes but is not limited to antibiotic resistance gene, herbicide resistance gene, fluorescent protein gene, etc. Specifically, the screening genes include, but are not limited to: chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.

The invention also discloses a method for improving the anthocyanin content of a plant, which can improve the anthocyanin content in the tissue organ of the plant by co-expressing the ThMYB1 or ThMYB2 gene provided by the invention and any bHLH transcription factor in the tissue organ of the plant.

The bHLH transcription factor can be isolated from any plant, including but not limited to the ThR 1and ThR2 genes provided by the invention, as well as the ZmR and ZmB genes from maize (Ahmed N, et al. Transmission expression of an anticancer in degrading maize by way of C1and B-peru regulatory genes for anticancer in synthesis. Breeding Sci.2003;53 (1): 29-34.).

The method for increasing the anthocyanin content of the plant can be used for increasing the anthocyanin content of any tissue or organ of the plant. Specifically, if one wants to increase the anthocyanin content in various tissues of a plant as a whole, the expression of both the ThMYB1 or ThMYB2 gene and the bHLH transcription factor can be promoted using a constitutive promoter. If only the anthocyanin content in a certain tissue or organ is to be increased, the ThMYB1 or ThMYB2 gene and the bHLH transcription factor can be promoted to be expressed by using a promoter specifically expressed in the tissue or organ.

The invention also discloses a method for improving the content of plant anthocyanin, and the method can improve the content of the plant anthocyanin in the plant tissue organ by co-expressing the ThR1 or ThR2 gene and any MYB transcription factor provided by the invention in the plant tissue organ.

The MYB transcription factor can be isolated from any plant, including but not limited to the ThMYB 1and ThMYB2 genes provided by the invention, and the ZmC1 gene from maize (Ahmed N, et al. Transmission expression of an anticancer in degrading maize B1 and B-peru regulation genes for anticancer in synthesis. Breeding Sci.2003;53 (1): 29-34.).

The method for increasing the anthocyanin content of the plant can be used for increasing the anthocyanin content of any tissue or organ of the plant. Specifically, if the anthocyanin content in each tissue of the plant is to be integrally increased, the ThR1 or ThR2 gene and the MYB transcription factor can be promoted to be expressed by using a constitutive promoter. If the anthocyanin content in a certain tissue or organ is only to be increased, the ThR1 or ThR2 gene and MYB transcription factors can be promoted to be expressed by using a promoter specifically expressed in the tissue or organ.

The present invention also provides a visual selection marker gene that produces a macroscopically blue selection marker in a tissue organ of a plant by co-expressing a ThMYB1 or ThMYB2 gene with either of the bHLH transcription factors. Or the ThR1 or ThR2 gene provided by the invention and any MYB transcription factor are co-expressed in the tissue organ of the plant, so that a macroscopic blue screening marker is generated in the tissue organ of the plant.

The screening marker gene disclosed by the invention can be used for distinguishing transgenic and non-transgenic materials.

Specifically, the screening marker gene provided by the invention can be used as a screening marker in the breeding process of a male sterile line, after the fertility restorer gene, the pollen inactivation gene and the screening marker gene provided by the invention are transferred into the male sterile line, the fertility restorer gene can restore the fertility of the male sterile line, the pollen inactivation gene can inactivate pollen containing a transformed exogenous gene, namely lose the insemination capability, the screening marker gene provided by the invention can be used for sorting transgenic seeds or tissues and non-transgenic seeds or tissues, the sorted non-transgenic seeds are used as sterile lines to produce hybrid seeds, and the transgenic seeds are used as maintainer lines to continuously and stably produce the sterile lines.

The screening marker gene provided by the invention can also be used as a screening marker in the reproduction process of a female sterile line, and the female fertility gene, the pollen inactivation gene and the screening marker gene provided by the invention are transferred into the female sterile line. The screening marker gene provided by the invention can be used for sorting transgenic seeds and non-transgenic seeds, the sorted non-transgenic seeds are used as female sterile lines to produce hybrid seeds, and the transgenic seeds are used as maintainer lines to continuously and stably produce female sterile lines and female sterile maintainer lines.

The invention also provides a promoter of the blue-grain gene, the promoter has the characteristic of aleurone layer specific expression, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 13, 14, 15 or 16. 13, 14, 15 or 16 of SEQ ID NO. is connected with a reporter gene GUS, a vector is constructed to transform rice and wheat, the expression activity and the expression pattern of GUS in a transgenic plant are detected and analyzed, and the GUS staining analysis is carried out on roots, stems, leaves, flowers and seeds of the transgenic plant, so that the result shows that the promoter provided by the invention drives the GUS gene to express in an aleurone layer of plant seeds. The blue particle gene promoter SEQ ID NO 13, 14, 15 or 16 provided by the invention is a promoter for aleurone layer specific expression.

The aleurone layer specific expression promoter provided by the invention contains a nucleotide sequence shown as SEQ ID NO 13, 14, 15 or 16 in a sequence table, or contains a nucleotide sequence with more than 90% of similarity with the nucleotide sequence shown as SEQ ID NO 13, 14, 15 or 16, or contains 500 and more than 500 continuous nucleotide fragments from the SEQ ID NO 13, 14, 15 or 16 sequence, and can drive the nucleotide sequence operatively connected with the promoter to be expressed in an aleurone layer of a plant seed. Expression vector, transgenic cell line, host bacteria and the like containing the sequence all belong to the protection scope of the invention. Primer pairs for amplifying any nucleotide fragment of the promoter of SEQ ID NO. 13, 14, 15 or 16 disclosed by the invention are also within the protection scope of the invention.

A "promoter" as used herein refers to a DNA regulatory region which typically contains a TATA box which directs RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. The promoter may also contain other recognition sequences, usually located upstream or 5' of the TATA box, often referred to as upstream promoter elements, which function to regulate transcription efficiency. It will be appreciated by those skilled in the art that while nucleotide sequences have been identified for the promoter regions disclosed herein, it is within the scope of the present invention to isolate and identify other regulatory elements in the region upstream of the TATA box of the particular promoter region identified herein. Thus, the promoter regions disclosed herein are typically further defined as comprising upstream regulatory elements, such as those used to regulate the function of tissue and temporal expression of coding sequences, enhancers, and the like. In the same manner, promoter elements that enable expression in a target tissue (e.g., male tissue) can be identified, isolated, and used with other core promoters to verify male tissue-preferred expression. Core promoter refers to the minimal sequence required to initiate transcription, such as the sequence known as the TATA box, which is commonly found in promoters of genes encoding proteins. Thus, the aleurone layer-specific expression promoters provided by the present invention may alternatively be used in association with their own or other source of core promoters. The core promoter may be any one of known core promoters, such as cauliflower mosaic virus 35S or 19S promoter (U.S. Pat. No.5,352,605), ubiquitin promoter (U.S. Pat. No.5,510,474), IN2 core promoter (U.S. Pat. No.5,364,780), or figwort mosaic virus promoter.

The function of the gene promoter of the invention can be analyzed by the following method: operably connecting a promoter sequence with a reporter gene to form a transformable vector, transferring the vector into a plant, and observing the expression condition of the reporter gene in each tissue organ of the plant to confirm the expression characteristic of the transgenic progeny; or subcloning the vector into an expression vector for transient expression experiment, and detecting the function of the promoter or the regulatory region thereof through the transient expression experiment.

The choice of an appropriate expression vector for testing the function of the promoter or regulatory region will depend on the host and the method of introducing the expression vector into the host, and such methods are well known to those of ordinary skill in the art. For eukaryotes, regions in the vector include regions that control transcription initiation and control processing. These regions are operably linked to a reporter gene comprising YFP, uid, GUS gene or luciferase. Expression vectors containing putative regulatory regions located in genomic fragments can be introduced into intact tissues, such as staged pollen, or into callus tissue for functional validation.

In addition, the nucleotide sequence of the aleurone layer-specific expression promoter provided by the present invention, or a fragment or variant thereof, may be assembled with a heterologous nucleotide sequence in an expression cassette for expression in a plant of interest, more specifically, in the seed of the plant. The expression cassette has appropriate restriction sites for insertion of the promoter and heterologous nucleotide sequence. These expression cassettes can be used to genetically manipulate any plant to obtain the desired corresponding phenotype.

The aleurone layer specific expression promoter disclosed by the invention can be used for driving the expression of genes, such as but not limited to genes related to yield increase, genes for improving the nutritional value of seeds, genes for improving the anthocyanin content, fluorescent protein genes and the like, so that transformed plants can obtain corresponding phenotypes.

The invention also provides an expression cassette, a vector or an engineering strain, wherein the expression cassette, the vector or the engineering strain comprises the aleurone layer specific expression promoter SEQ ID NO. 13, 14, 15 or 16 provided by the invention, or comprises 500 and more than 500 continuous nucleotide fragments derived from the SEQ ID NO. 13, 14, 15 or 16 sequence.

The aleurone layer specific expression promoter provided by the invention can be used for specific expression of an exogenous gene in seeds, so that adverse effects caused by continuous expression of the exogenous gene in other tissues of plants are avoided, and the aleurone layer specific expression promoter has an important application value in plant genetic engineering research.

The nucleotide sequence and promoter sequence or expression cassette of the blue-particle gene provided by the present invention can be inserted into a vector, plasmid, yeast artificial chromosome, bacterial artificial chromosome or any other vector suitable for transformation into a host cell. Preferred host cells are bacterial cells, in particular for cloning or storing polynucleotides, or for transforming plant cells, such as E.coli, agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or vector may be inserted into the genome of the transformed plant cell. The insertions may be localized or random insertions.

The nucleotide sequence, the vector or the expression cassette is transferred into a plant or introduced into the plant or transformed into the plant, and the nucleotide sequence, the vector or the expression cassette is transferred into a receptor cell or a receptor plant by a conventional transgenic method. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform the recombinant expression vector into a plant cell to produce a transgenic plant of the invention. The transformation methods may include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection. The transformation method also includes Agrobacterium-mediated plant transformation methods and the like.

Compared with the prior art, the invention has the following beneficial effects: the invention provides a blue particle gene and a promoter thereof, wherein the blue particle gene can improve the content of anthocyanin in plants, and because the anthocyanin has an anti-oxidation function, the discovery of the anthocyanin synthesis related gene undoubtedly increases the nutritional value and medical value of edible parts of the plants in the times that people increasingly pursue healthy life due to aggravated environmental pollution. Meanwhile, the blue granule gene can be used as a screening marker while improving the anthocyanin content of the plant, so that the screening marker elimination process in the transgenic process is avoided, the time and steps of transgenic bioengineering are saved, and the blue granule gene has great application value in practical production application.

Reference to the literature

Zhou K,Wang S,Feng Y,Liu Z,Wang G.The 4E-system of producing hybrid wheat.Crop Sci.2006；46(1):250-255.

Plum tremble, mummy, blue grain single wheat study (one) [ J ]. Genetics report, 1982 (6): 15.

Zheng Q.,Li B.,Mu S.,Zhou H.,Li Z.(2006).Physical mapping of the blue-grained gene(s)from Thinopyrum ponticum by GISH and FISH in a set of translocation lines with different seed colors in wheat.Genome 49,1109-1114.

Dubcovsky,J.,Luo,M.C.,Zhong,G.Y.,Bransteitter,R.,Desai,A.,Kilian,A.,et al.(1996).Genetic map of diploid wheat,Triticum monococcum L.,and its comparison with maps of Hordeum vulgare L.Genetics 143,983-999.

Himi,E.,and Noda,K.(2005).Red grain colour gene(R)of wheat is a Myb-type transcription factor.Euphytica 143,239-242.

Khlestkina,E.K.Genes determining coloration of different organs in wheat.Russ.J.Genet.Appl.

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Shoeva,O.Y.,Gordeeva,E.L.,and Khlestkina,E.K.(2014).The regulation of anthocyanin synthesis in the wheat pericarp.Molecules 19,20266-20279.

"Gongxiao", xuejing, zhang Xiaodong "2011" research on regulatory genes in the synthetic pathway of plant anthocyanidin, progress of biotechnology 1 (6): 381-390

Ahmed N,Maekawa M,Utsugi S,Himi E,Ablet H,Rikiishi K,et al.Transient expression of anthocyanin in developing wheat coleoptile by maize C1and B-peru regulatory genes for anthocyanin synthesis.Breeding Sci.2003；53(1):29-34.

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

Drawings

Fig. 1 is the kernel color of blue and white wheat. The left panel is the deep blue grain of blue grain wheat 3114BB, the right panel is the white grain of its parent white grain wheat 3114.

FIG. 2 is a genomic PCR used to verify the source of the four blue grain genes ThMYB1, thMYB2, thR 1and ThR2. The left panel is an agarose gel electrophoresis of the PCR products of the ThMYB 1and ThMYB2 genes, and the right panel is an agarose gel electrophoresis of the PCR products of the ThR 1and ThR2 genes. No expression of four genes could be detected in the genome of white grain wheat 3114, but in the genomes of blue grain wheat 3114BB and elytrigia elongata.

FIG. 3 is a graph demonstrating the source of four blue granule genes using semi-quantitative RT-PCR. Agarose gel electrophoresis of the PCR products showed that no expression of four genes could be detected in the cDNA of white grain wheat 3114, but four genes could be detected in the cDNA of blue grain wheat 3114BB, where ACTIN is the housekeeping protein gene.

FIG. 4 is a graph showing the expression pattern of four blue grain genes in blue grain wheat 3114BB by semi-quantitative RT-PCR. The left panel is agarose gel electrophoresis of the PCR products of the blue-particle gene in different plant organs or tissues, and the right panel is agarose gel electrophoresis of the PCR products of the blue-particle gene at various developmental stages of the aleurone layer, wherein ACTIN is the housekeeping protein gene. The expression of four genes can not be detected in cDNA of root, stem, leaf, embryo and endosperm, but high expression of four genes can be detected in aleurone layer, and the expression quantity of different blue granule genes in different development days of aleurone layer is not identical. DPA refers to days post anthesis, i.e. "days post anthesis".

FIG. 5 is a gene gun transient transformation experiment to verify that four blue grain genes can induce wheat coleoptile to produce red anthocyanin spots. Combining the blue grain genes pairwise, co-transforming the combined blue grain genes into wheat coleoptiles respectively, culturing the wheat coleoptiles in a light incubator for 16 hours, and observing the wheat coleoptiles by using a microscope to find that the combination of ThMYB1+ ThR 1and the combination of ThMYB2+ ThR1 can induce most cells to generate red anthocyanin spots, the combination of ThMYB1+ ThR2 can only induce a few cells to generate anthocyanin, and the combination of ThMYB2+ ThR2 can not induce the generation of anthocyanin.

FIG. 6 is a schematic diagram of the construction of plant transformation vectors for wheat transgenic experiments. Wherein LB and RB are the left and right borders of the T-DNA, respectively; the expression of the Bar resistance gene is driven by Ubip (the promoter of the Ubi gene), and is terminated by the Nos terminator; expression of the ThMYB 1and ThR1 genes is regulated by respective promoters and terminators.

FIG. 7 is T of ThMYB1+ ThR1 transgenic wheat in the background of the Agrobacterium-mediated obtention of wheat variety fielder ₁ The seed color of the plant is substituted. The left panel shows white grains of the non-transgenic wheat variety fielder, the middle panel shows T ₁ The right picture is T ₁ And generating a strain with light blue grains in the transgenic plant.

Detailed Description

The following examples are given for the detailed implementation and the specific operation procedures, but the scope of the present invention is not limited to the following examples.

Example 1 cloning of blue-grain Gene

In order to clone the blue grain gene from the 4Ag chromosome of elytrigia elongata, the differential expression analysis of blue grain and white grain wheat is carried out (see figure 1), and theoretically, the differential expression genes of the blue grain and the white grain wheat comprise two types: 1) The gene expressed by the 4Ag chromosome of the elytrigia elongata comprises the blue grain gene which is wanted to be cloned by the invention; 2) Downstream gene changes caused by the expression of elytrigia elongata 4Ag chromosome, and the genes are from wheat genome. The present invention intends to obtain the blue granule gene described in 1) from the analysis thereof. The blue-kernel wheat 3114BB and the parent white-kernel wheat 3114 are selected as materials, because the aleurone layer of wheat grains is blue, the aleurone layer of the wheat grains is changed into blue after about 20 days of flowering, and the aleurone layer of the wheat grains is changed into blue after about 25 days, the aleurone layer of the wheat grains is stripped, the blue-kernel and white-kernel materials are respectively 2 parts which are respectively marked as blue 1, blue 2, white 1and white 2, RNA is extracted, high-throughput sequencing (PE 125) is carried out, and the data amount of 9G is respectively measured on each sample.

Since the published wheat reference genome only covers 61% of the whole wheat genome and the integrity of gene annotation and assembly sequences (scaffolds) are fragmented, direct sequence alignment and differential gene expression analysis cannot find the target gene. The invention uses a three-step elimination method, firstly eliminates a double-end sequence which is accurately compared to a wheat reference genome, secondly eliminates a high-expression gene in white-grain wheat in a residual double-end short-sequence recombinant gene, and finally eliminates a gene which is irrelevant to anthocyanin metabolism in a differential expression gene which meets the conditions.

Through the analysis, 139 differentially expressed genes on non-wheat reference genomes are obtained, wherein 35 genes are highly expressed in blue-grain wheat and hardly expressed in white-grain wheat, and only two target genes are used for respectively coding MYB protein and bHLH protein after gene function annotation analysis is carried out to exclude genes irrelevant to anthocyanin metabolism, and the genes are named as ThMYB 1and ThR1. Based on the known cDNA sequences, the coding region genome sequences of the two genes are obtained by PCR amplification, and in the process, the invention finds that whether ThMYB1 or ThR1 is used, another sequence which is highly homologous with the ThMYB1 or ThR1 is obtained by PCR amplification, so that the invention clones the two homologous sequences and respectively names ThMYB2 and ThR2. Through chromosome walking technology, the invention respectively obtains the promoter sequence and the terminator sequence of the 4 genes.

Wherein, the genome nucleotide sequence from the start codon to the stop codon of the ThMYB1 gene is shown as SEQ ID NO. 1, the nucleotide sequence of the coding region (CDS) is shown as SEQ ID NO. 2, the amino acid sequence coded by the CDS is shown as SEQ ID NO. 3, the promoter sequence is shown as SEQ ID NO. 13, and the terminator sequence is shown as SEQ ID NO. 17. Wherein the genome nucleotide sequence from the start codon to the stop codon of the ThMYB2 gene is shown as SEQ ID NO. 4, the nucleotide sequence of a coding region (CDS) of the ThMYB2 gene is shown as SEQ ID NO.5, the amino acid sequence coded by the CDS of the ThMYB2 gene is shown as SEQ ID NO. 6, the promoter sequence of the CDS of the ThMYB gene is shown as SEQ ID NO. 14, and the terminator sequence of the CDS of the ThMYB2 gene is shown as SEQ ID NO. 18. Wherein the genome nucleotide sequence from the start codon to the stop codon of the ThR1 gene is shown as SEQ ID NO. 7, the nucleotide sequence of the coding region (CDS) is shown as SEQ ID NO. 8, the amino acid sequence coded by the CDS is shown as SEQ ID NO. 9, the promoter sequence is shown as SEQ ID NO. 15, and the terminator sequence is shown as SEQ ID NO. 19. Wherein, the genome nucleotide sequence from the start codon to the stop codon of the ThR2 gene is shown as SEQ ID NO. 10, the nucleotide sequence of a coding region (CDS) thereof is shown as SEQ ID NO. 11, the amino acid sequence coded by the CDS thereof is shown as SEQ ID NO. 12, the promoter sequence thereof is shown as SEQ ID NO. 16, and the terminator sequence thereof is shown as SEQ ID NO. 20.

The invention verifies the source of the 4 genes through genome PCR and semi-quantitative RT-PCR. Genomic PCR showed that: in white-grain wheat 3114, the amplification was less than that of ThMYB1, thMYB2, thR 1and ThR2, while in blue-grain wheat 3114BB and Elytrigia elongata genomes, the amplification could be applied to these 4 genes, indicating that these four genes are indeed from Elytrigia elongata 4Ag chromosome rather than from common wheat (see FIG. 2). Semi-quantitative RT-PCR results in aleurone layer tissue cDNA 25 days after flowering also showed: thMYB1, thMYB2, thR1, and ThR2 are not expressed in the white grain wheat aleurone layer but expressed only in the blue grain wheat aleurone layer at high levels (see fig. 3). The results show that the four genes of ThMYB1, thMYB2, thR 1and ThR2 are all from the 4Ag chromosome of elytrigia elongata and are expressed in a high amount in the aleurone layer of the blue-grain wheat, and are probably the blue-grain genes searched by the invention.

Example 2 expression Pattern of the blue granule Gene

The invention verifies the expression pattern of the 4 genes by using semi-quantitative RT-PCR. The results are shown in FIG. 4. Firstly, the expression of 4 genes can not be detected in roots, stems and leaves of a blue-grain wheat 3114BB vegetative organ, and 4 genes can be specifically expressed in aleurone layer tissues in different tissues of seeds 20 days (20 DPA) after flowering, but can not be detected in embryo and endosperm tissues, which indicates that the 4 genes are all genes specifically expressed in aleurone layer tissues, and promoters of the genes are all promoters for driving the genes to be specifically expressed in aleurone layers. Further, the expression patterns of ThMYB1, thMYB2, thR 1and ThR2 in the aleurone layer of the blue-grain wheat seeds on different days after flowering are analyzed, and the expression patterns of 2 MYB genes and 2 bHLH genes are different: thMYB 1and ThMYB2 were expressed at very low levels in the aleurone layers of 10DPA and 15DPA, with a sudden high expression at 20DPA, and thereafter gradually decreased at 25DPA and 30 DPA; thR 1and ThR2 were not detected in the 10DPA and 15DPA aleurone layers, and were expressed in high amounts starting from 20DPA to 30 DPA. The above results show that ThMYB1, thMYB2, thR1, and ThR2 are all wheat aleurone layer-specific expression genes, and their expression is spatiotemporal specific.

The promoter sequences SEQ ID NO 13, 14, 15 and 16 of the 4 genes drive GUS genes to perform function verification in plants such as rice, corn and the like, and the results show that the promoters drive GUS to be specifically expressed in an aleurone layer, so that the promoters provided by the invention are aleurone layer specific expression promoters.

Example 3 Gene gun transient transformation of wheat coleoptile experiment

In previous studies, maize MYB family transcription factor C1and bHLH family transcription factor B1 were co-transiently transformed into wheat coleoptiles using particle gun bombardment, which induced red anthocyanin spots in wheat coleoptiles (Ahmed N, maekawa M, utsugi S, himi E, ablet H, rikishi K, et al. Transmission expression of antisense in transforming wheat coleoptile by domain C1and B-expert regulated genes for antisense in synthesis. Breeding Sci.2003;53 (1): 29-34). In order to verify whether the 2 MYB genes and the 2 bHLH genes obtained by the invention have the same functions, the 4 genes are used for constructing a gene gun transient transformation vector. Firstly, cloning an NOS terminator into pEASY-T1simple (all-purpose gold company) vectors, then inserting coding frames of 4 genes of ThMYB1, thMYB2, thR 1and ThR2 before NOS, and finally inserting a Ubi (Ubiquitin) promoter from corn before the coding frame of the gene by using an in-fusion method to drive the gene expression to form four vectors of Ubi:: thMYB1, ubi:: thMYB2, ubi:: thR 1and Ubi:: thR2. Experimental reference (Ahmed et al, 2003), the transformation vectors for MYB and bHLH genes were combined in pairs, i.e., four combinations of ThMYB1+ ThR1, thMYB1+ ThR2, thMYB2+ ThR 1and ThMYB2+ ThR2 were co-transformed into wheat coleoptiles, and after 16 hours of culture in a light incubator, observed under a microscope and photographed, the results are shown in FIG. 5: the combination of ThMYB1+ ThR 1and ThMYB2+ ThR1 is capable of inducing the production of red anthocyanins, and the combination of ThMYB1+ ThR2 is also capable of inducing the production of anthocyanins by cells.

Example 4 construction of plant expression vectors

In order to further verify that ThMYB1, thMYB2, thR 1and ThR2 are blue grain genes, the stable transformation vector is constructed by selecting the ThMYB1 gene and the ThR1 gene and is used for a wheat transgenic experiment. A binary expression vector pCAMBIA1300 is taken as a framework, and a hygromycin resistance expression frame driven by a plant resistance screening gene 35S promoter on the pCAMBIA1300 is replaced by a Bar resistance gene expression frame driven by a Ubi promoter on a pAHC20 vector. On the basis, the present invention inserts a 3215bp ThMYB1 genomic sequence (comprising 1952bp promoter sequence, 822bp genomic sequence and 441bp terminator sequence) and a 4422bp ThR1 genomic sequence (comprising 2084bp promoter sequence, 1720bp CDS sequence and 618bp terminator sequence) into a multiple cloning site, thereby forming a plant expression vector (see fig. 6).

Example 5 obtaining of transgenic blue-grain wheat

The plant expression vector constructed in example 4 was transformed into Agrobacterium strain C58C1 by the electroporation method. Transferring the vector into wheat variety fielder by utilizing an agrobacterium-mediated wheat transformation system to obtain 96 transgenic positive T ₀ And (5) plant generation. T is ₀ Plant generation plantThe color of wheat grains is observed during seed harvest, and some strains have deep blue grain separation and some strains have light blue grain separation (about 34 percent), and the specific results are shown in a figure 7. The transgenic result shows that the ThMYB 1and ThR1 genes are co-expressed in a plant, the anthocyanin content in the plant can be increased, and the ThMYB1, thMYB2, thR 1and ThR2 provided by the invention are blue grain genes in wheat.

Example 6 functional verification of blue-grain genes in Rice, arabidopsis, maize plants

The ThMYB1 or ThMYB2 gene provided by the invention, the ThR1, thR2, zmR and ZmB genes are combined in pairs in a mode of adding one MYB gene and one HLH gene, and the combined gene is transferred into plants such as rice, arabidopsis thaliana and corn for co-expression, so that the same function of improving the anthocyanin content in the plants is found.

Sequence listing

<110> university of capital education

Unknown Xingwang System Crop Design Frontier Laboratory (Beijing) Co., Ltd.

<120> wheat blue grain gene and application thereof

<160> 20

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 1

atggcgaagg aaggggtgaa gagaggggca tggactggca aggaggacga aaccttggct 60

tcctacgtca aggcgcatgg cgaaggcaga tggaacgaag tccctcagag agctggtaag 120

tccctagcta ggtaaacaga atcaatctag agaatggagt gctaaacgta atttacaggt 180

cttcggcggt gcggcaagag ctgtcggctg cggtggctga actacctccg gccgaacatc 240

aagcggggaa acatatccaa tgatgaggag gagatcatcg tcaggctcca cgctctcctt 300

ggcaacaggt ggtcgctcat cgctggcagg ttgcctggcc gaacagacaa cgaaatcaag 360

aactactgga acagcaccct tggccggaag gtgcttcccg caccacattc cgccaccagg 420

atggttgcca cgccagacac ctccgccggc tccggatctt acaaagaggc gtcggcaagt 480

ctgtcaagct ctggacctgg tacaagcgac aaggctgcga cgccgtcgcc gctgccggcc 540

acgctgtggg cgccaaagcc tgtgaggcac acggggcacc ccttcttcct ccgggatagg 600

ccgccgccct tgccggttgc ggagacgcga accgtggcca acggggatgc ctgcagcggc 660

agcagctcgg tgacatcgga gttcccggct gtaccgccct cgttaggcag cgacgactgg 720

atggacgaag tgagagcctt ggaatcgttt ctcgagtccg acgaagactg ggtaaactct 780

gtggagatgc caacgcccaa ctctcacaca tcatcgagtt aa 822

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<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

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atggcgaagg aaggggtgaa gagaggggca tggactggca aggaggacga aaccttggct 60

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cggcggtgcg gcaagagctg tcggctgcgg tggctgaact acctccggcc gaacatcaag 180

cggggaaaca tatccaatga tgaggaggag atcatcgtca ggctccacgc tctccttggc 240

aacaggtggt cgctcatcgc tggcaggttg cctggccgaa cagacaacga aatcaagaac 300

tactggaaca gcacccttgg ccggaaggtg cttcccgcac cacattccgc caccaggatg 360

gttgccacgc cagacacctc cgccggctcc ggatcttaca aagaggcgtc ggcaagtctg 420

tcaagctctg gacctggtac aagcgacaag gctgcgacgc cgtcgccgct gccggccacg 480

ctgtgggcgc caaagcctgt gaggcacacg gggcacccct tcttcctccg ggataggccg 540

ccgcccttgc cggttgcgga gacgcgaacc gtggccaacg gggatgcctg cagcggcagc 600

agctcggtga catcggagtt cccggctgta ccgccctcgt taggcagcga cgactggatg 660

gacgaagtga gagccttgga atcgtttctc gagtccgacg aagactgggt aaactctgtg 720

gagatgccaa cgcccaactc tcacacatca tcgagttaa 759

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<211> 252

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<213> Elytrigia elongata (Elytrigia elongata)

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Met Ala Lys Glu Gly Val Lys Arg Gly Ala Trp Thr Gly Lys Glu Asp

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Glu Thr Leu Ala Ser Tyr Val Lys Ala His Gly Glu Gly Arg Trp Asn

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Glu Val Pro Gln Arg Ala Gly Leu Arg Arg Cys Gly Lys Ser Cys Arg

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Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn Ile Lys Arg Gly Asn Ile

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Ser Asn Asp Glu Glu Glu Ile Ile Val Arg Leu His Ala Leu Leu Gly

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Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro Gly Arg Thr Asp Asn

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Glu Ile Lys Asn Tyr Trp Asn Ser Thr Leu Gly Arg Lys Val Leu Pro

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Ala Pro His Ser Ala Thr Arg Met Val Ala Thr Pro Asp Thr Ser Ala

115 120 125

Gly Ser Gly Ser Tyr Lys Glu Ala Ser Ala Ser Leu Ser Ser Ser Gly

130 135 140

Pro Gly Thr Ser Asp Lys Ala Ala Thr Pro Ser Pro Leu Pro Ala Thr

145 150 155 160

Leu Trp Ala Pro Lys Pro Val Arg His Thr Gly His Pro Phe Phe Leu

165 170 175

Arg Asp Arg Pro Pro Pro Leu Pro Val Ala Glu Thr Arg Thr Val Ala

180 185 190

Asn Gly Asp Ala Cys Ser Gly Ser Ser Ser Val Thr Ser Glu Phe Pro

195 200 205

Ala Val Pro Pro Ser Leu Gly Ser Asp Asp Trp Met Asp Glu Val Arg

210 215 220

Ala Leu Glu Ser Phe Leu Glu Ser Asp Glu Asp Trp Val Asn Ser Val

225 230 235 240

Glu Met Pro Thr Pro Asn Ser His Thr Ser Ser Ser

245 250

<210> 4

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<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

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atggcgaagg aagtggtgaa gagaggggca tggactggca aggaggacga aaccttggct 60

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tccctagctg ggtaaacaga atcaatctag acaatggagt gctaaacgta atttacaggt 180

cttcggcggc gcggcaagag ctgtcggctg cggtggctga actacctccg gccgaacatc 240

aagcggggaa acatatcaaa cgatgaggag gagatcatcg tcaggctcca cgctctcctt 300

ggcaacaggt ggtcgctcat cgctggcagg ttgcccggcc gaacagacaa cgaaatcaag 360

aactactgga acagcaccct tggccggaag gtgcttcccg caccacattc cgccaccagg 420

atggttgcca cgccagacac ctccgccgcc tccggatctt acacagaggc gtcggcaagt 480

ctatctagct ctggacctgg tacaagcgac aaggctgcga cgccgtcgcc gctcccggcc 540

acgctgtggg cgccaaagcc tgtgaggtac acgggccacc ccttcttcct ccgggatagg 600

ccgcggccct tgccggttgc ggagacgcga accgtggcca acggggatgc ctacagcggc 660

agcagctcgg tgacatcgga gttcccggct gtcccgccct cgttaggcag tgacgactgg 720

atggacgaag taagagcctt ggaatcgttt ctcgagtccg acgaagactg ggtaaactct 780

gtggagatgc cggattaa 798

<210> 5

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<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

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atggcgaagg aagtggtgaa gagaggggca tggactggca aggaggacga aaccttggct 60

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cggcggcgcg gcaagagctg tcggctgcgg tggctgaact acctccggcc gaacatcaag 180

cggggaaaca tatcaaacga tgaggaggag atcatcgtca ggctccacgc tctccttggc 240

aacaggtggt cgctcatcgc tggcaggttg cccggccgaa cagacaacga aatcaagaac 300

tactggaaca gcacccttgg ccggaaggtg cttcccgcac cacattccgc caccaggatg 360

gttgccacgc cagacacctc cgccgcctcc ggatcttaca cagaggcgtc ggcaagtcta 420

tctagctctg gacctggtac aagcgacaag gctgcgacgc cgtcgccgct cccggccacg 480

ctgtgggcgc caaagcctgt gaggtacacg ggccacccct tcttcctccg ggataggccg 540

cggcccttgc cggttgcgga gacgcgaacc gtggccaacg gggatgccta cagcggcagc 600

agctcggtga catcggagtt cccggctgtc ccgccctcgt taggcagtga cgactggatg 660

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Met Ala Lys Glu Val Val Lys Arg Gly Ala Trp Thr Gly Lys Glu Asp

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Glu Thr Leu Ala Ser Tyr Val Lys Ala His Gly Glu Gly Arg Trp Asn

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Glu Val Pro Gln Arg Ala Gly Leu Arg Arg Arg Gly Lys Ser Cys Arg

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Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn Ile Lys Arg Gly Asn Ile

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Ser Asn Asp Glu Glu Glu Ile Ile Val Arg Leu His Ala Leu Leu Gly

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Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro Gly Arg Thr Asp Asn

85 90 95

Glu Ile Lys Asn Tyr Trp Asn Ser Thr Leu Gly Arg Lys Val Leu Pro

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Ala Pro His Ser Ala Thr Arg Met Val Ala Thr Pro Asp Thr Ser Ala

115 120 125

Ala Ser Gly Ser Tyr Thr Glu Ala Ser Ala Ser Leu Ser Ser Ser Gly

130 135 140

Pro Gly Thr Ser Asp Lys Ala Ala Thr Pro Ser Pro Leu Pro Ala Thr

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Leu Trp Ala Pro Lys Pro Val Arg Tyr Thr Gly His Pro Phe Phe Leu

165 170 175

Arg Asp Arg Pro Arg Pro Leu Pro Val Ala Glu Thr Arg Thr Val Ala

180 185 190

Asn Gly Asp Ala Tyr Ser Gly Ser Ser Ser Val Thr Ser Glu Phe Pro

195 200 205

Ala Val Pro Pro Ser Leu Gly Ser Asp Asp Trp Met Asp Glu Val Arg

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Ala Leu Glu Ser Phe Leu Glu Ser Asp Glu Asp Trp Val Asn Ser Val

225 230 235 240

Glu Met Pro Asp

<210> 7

<211> 12351

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

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atggcgctat cagctcctcc cagtcaggaa cagccgtcgg ggaagcaatt cggctaccag 60

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agcccgcgcc caggccactc ctcgtaatga ataaatgacc actctctatc tctatattat 180

gtatcatatt tgggtttgta gcaaacccac aaaattccta cttttcatat atatagtgtc 240

cgtcgtcgac gccgccggtt cgttttgcag agttctggcg tggaaggatg ggttctacaa 300

cggcgagata aagactagaa agattaccgg ctcgaccact acggagctta cagcggacga 360

gcgcgtcatg cacagaagca agcaactgag ggagctctac gaatcgctct tgcccggcaa 420

ctccaacaac cgggcaaggc gaccaaccgc ctcactgtca ccggaggatc tcggggacgg 480

cgagtggtat tacaccataa gcatgactta caccttccac cctaatcaag ggtacgtact 540

tatcaaccat gatggatgtg atatatgtgc ctgccatgtt tcataccgtc catattttta 600

tatacgggta catttttttt tttgcgggtt tatatacggg tacatgacaa agacaatttt 660

tgcggttagt tatcttgtta atatatactg tcatgttgcc gccaggaaaa gatcacttca 720

agaaaattat aaacaaacat atataatacg tccaattatt ttcagccttt tccttttttt 780

aaattaaata catagagtcc aagcttcaaa tcctgcactt aaacctcaag ctaatctgtt 840

acactccctt gcagctagtc tcttactagt attatctctt cttctttttt tagaaaaact 900

ttcgatctat tcatcatgac agtaaaaaaa aaacactaga aataacaaaa attacattca 960

tatttataga acacctagcg acgattgcaa acattcaact gtgcgccgaa ggcgcactac 1020

actcatcgcc ccttcctggt tggagttggg caaacttcat tgtagtagac ggtaggaaag 1080

tctttgtgct aaggccttaa aggaccagca caccatgaca acaaccatca tcattaaaga 1140

aaagcatagg tcgaaaggat tcaacaagta gacacataac acaggaaaac taaggccgaa 1200

tccaggtata tccattgaag tcaaacactg accgaatccc gcgagattca ccgacatgcc 1260

ctccgacaat gctagatgca tcaacgggat agggactttt ttttagaaag gaggattacc 1320

cccggcttct gcatctggga gatgcatgca gccattgtat taattattca caaagacctt 1380

acaaaatagt acatcaggta gtctgaagcc atcatcttat caacaactgt cgctactcct 1440

atccacttga tgaaatggtg ccgatagtcc gagcctaata ccagacagac atcgcaccaa 1500

agcctaacat ctaaagccga gggcccaacc aaacgggata gggacttggc agggtgaacc 1560

ttattttatc ttcagagagc agagcgttgc ctcacggcct catcttcctg agcagcacac 1620

aaacactaaa taaaactaaa aaaactctta aaatgaagta ggagccctct cgccggcaag 1680

agccgggatc catcatacct ccatggccct aaagccgaag gaggcgagga agctcgacgg 1740

cagcgtcggt gggaggcgaa aagccctcgt ttcctgggag ttcgctcgtg gagcactaat 1800

actatcttga tactagctac cgtagactgc agtgttaaga tgtacatact gaagtgattt 1860

ttttctgtcg gcgtgaatgt acttactgcc aggttgccag gcaaaagctt tgcgagcaat 1920

caacatgttt ggctgtacaa cgctcaatac gcaaacacca gagttttccc ccgcgcgctc 1980

ttagcaaagg tatatctatt ctacagtact tatctattat gtgtgtatag caatgatgcg 2040

ggtcgggttt tcaacctcct tttctgaaat aagaaaacat cgtttggtct ttaactaata 2100

ataataatac tccaaaacgt gcatgatgtt gcccacacat ccgatggtca aatcagactg 2160

cttctattca ggtcagcaca ttacttccta ttatagatag atgggataca tacatttttc 2220

gtccttaaat tcttgtgaaa gtttacaaat cgtccctcaa ctcaaaacca tcaaaattca 2280

aaactcagtc cctcaactat taaaaccgga tatttttcgt ccttgataac gcttcaggcg 2340

gttttagtct gacatggaca ccggttttga ctaatatcgg ccacgtggcc tgggtttgac 2400

catcacgtca gtcagttaag tgactaaagg acctcctgtc gaagagaata aagggcagta 2460

tggcgcaggg ccatgcagct tcaggttcga tccaggtcac ctagttcttg tcatagatga 2520

cgttcgagca gtccaccagc gtagggaagc acgactgcgc cgccagcggg tgcagaagtc 2580

gaagggcgac ttgggccgcg gctgccgcag ggtctgacgt tagtcggtag caagtgctcg 2640

aagtcgaagg gcggcaggag gaaaccgatg ccggcgccgt cgccgacctg ccactcggcc 2700

ttggcgctgg tgaggacgga ggcatgcacc aagctcagga ggacggaggc gtccatggcg 2760

cctagcccat gtcacggtgc gccatggccg cgagctcggc cggcctcctg caccacagcc 2820

gtgtatgccc aaggccgcag cctcgcttgt gcgcgctcgt gcagccccac gcgcttcctc 2880

ctcgtccgct cccccatgcc cggccgtgct ccgagcgagc acccaccgcg ccctggccct 2940

gtcccgtgcc tgcgcaaccg cgtgttgacc ggccattgcc aggtgggccg accagtttac 3000

ttagctgatg gttttgacca ccagactaaa accgggccac atcggcagca tttgtcaaaa 3060

ccgggtatcc atgtcagact aaaaccgccc aaaactttac gaaggacgaa aaatattcag 3120

ttttaatagt tgagggactg agttttgatg gttttaaagt tgagggataa tttataaact 3180

ttgacagaag tttaaggacg aaaaatatac ttatccctag atagatacat agacgcacac 3240

atatacacat ctctgatctc aaaccgttca catttttcca gacaatcgtt tgcattccct 3300

tcatgggcgg tgtgcttgag ctcggaacgt cggatcaggt cggtgcacat gagtttcaat 3360

ttatgttctc agtaactctc agcatgtttc tccgacttgc cgacgggttc aacggtaacc 3420

ttttaatatt aggggtagct attttgtgtg ccaatacatg ctactagcta tttttgtatg 3480

tgttgtccca gtctaccatt ctgccccttt gcatttatgg cgttattcct ttctagtaca 3540

ttaatttaca ctagtcttcc actgcgtttt tctactacta cgtattttat actatacttg 3600

tgattatctt tagagcaagt ataatagagt gatgtaggca ggctataaga gatgtcacat 3660

cagattatgc ctagttggag gagagagaaa atgagagaga aaagaagcag gttacaagct 3720

tacggtcggc tgtagcacga gactcaacaa cctttgtgtg tgagagaggt gggccatata 3780

ttaattgtat gacaaactag tatgactagc tattgtacga gtgggctatt aggctgacta 3840

taagtgacat gacaactcta tatagacggc tcttggctat actattaacc atgctcttac 3900

ctctctcccg ccgccatgat gagagcctca ccggccctgt tactgggtca agggagacca 3960

acgcccaccg agcccgacca atcttgcagc ccacaccact acgtcaccct agcaactccg 4020

ctagtagccc gtcacctagg cacaatgact tcaacacctc gagagcaagc gccctgtcgc 4080

ggagccaccg tgctcgcaca tgtcgggatc tctcccacct tcgaagaggc aagggggcgc 4140

aacccatcat aatcctacgg cggcagcggc ggccaggttg ggcattggag ggggtgctgc 4200

ttagcatcgg agacatggtg tcgcctagtg gtacgacacg acccggagag gaaaacagaa 4260

aacattgcga gcgcaagttc gccacgagtg ttgcgtgtta tttttactaa ataacccttg 4320

aggatcttat ttttgtccac tgcccccaaa acgtatttcg tggcctgacc cctacgacca 4380

cgccaaataa tctggcgttt tagatacact agatgccacc ggtcaatctg acagcaataa 4440

attcaatgtg acagtggcgc ctacatgaca agaaaagtca aaagccaaat ttgaaggcct 4500

atatcatccg ataaaatctc cggttagtat ggcggggcat gctgcttcct tcttcctctg 4560

cacgaaccct gcgaggccac cggccaccgc caccgctggt agcagccggc tccagccacg 4620

gtagaggggc cctgcggtgg aggaagtcga cgggccggtg catgctctcc gccactgccg 4680

tgaccaccag catgaccgcc tcctcgtcct cgtccaccgc cgcgaccggc gtcgagcacg 4740

gggccgacgc gtcgtctgac gtattcccgc gcctctggaa ctctgaggtt ggggccacct 4800

tccgccgcac gtccgtggac ccggcggcac gcgacgagtc cgcggcacgg gaaggggcac 4860

tctcagcagc ggcgccattt ccaccgcatg gggacgactc atcggtggtg aggtcgccgc 4920

cggcacgctt cgacgcaagc agctcgagcg tcgagtccac attaacgcca ttgcacgaat 4980

ctgtagacgg tccctccacg ccgagtccga gcgcgcgccc gtcagccctt ccgcccgacc 5040

ccgtccaagt ccccattggt ggggcaagca actgcttggc cggtgctccc aacagcgagg 5100

atgacctcca cctagctcgg acgcgacgtc gccggccacc actgtaagat atggccgggg 5160

aagtagctgc cttatcgaag gagcccgcgg cggaccagtg gcagtgtggt gcaccgggac 5220

caacagaggg gggcagataa ccaaataagt tttttgagga gttagtgcac gaaataagat 5280

cctttagagt caattagtaa attaacacat tgttaatttt attaattctc gtattaattt 5340

tactaattga cacgagtgtc ttcctaaaaa aaaaatttaa cacgagtgtt gcaccgcgac 5400

gtgcaaccaa ccgtttgatt gcttctcgtt gccgtgagta tgcttgtgaa cgtcgctcgt 5460

aacgtgccac gggctaacgt ttgcactcca ccatctgtta acctttgatt accatttttt 5520

acggggtaaa acattgcatt actcaagagg aataagttct tcgctgcaga gagtagtagg 5580

gtcgtttaca agacctgaac atatgtgtgc tgtcatgcaa cactttattt attaggttgt 5640

agacttatct tgtcttgata tgtgtgatgt tacaataact agctatgtta caacatgccc 5700

ctctttcctc attaattagt cgccacatca tctgttttgc ctacagatgt gtgatgttac 5760

cacctatgtt actcccacta tgggtagtct tattgtgatt agcaccggtg tgccaacact 5820

tagtcccttt ccatgaggaa cccctcccag acggcctcat tgaaggatat ttgtccgtct 5880

tgtactcaat ggtgacgggc cctgctcaag ccgtacacca acaaggccta tccgcgggat 5940

gttcaggccg gaaatcacat tcataagtaa tttcgaatct ctcccgtgga ttaaaataaa 6000

aaagagcaca caatgttagg catcgccata tagtgttatg cacaaaaaaa aggaatacta 6060

aaaaacgtac catagcatgt gtgacgatgt ttgtcttagt caaaaggtgc ataaagggca 6120

cccccgtgta gtcatcagcg ggtgatagga tgtcgaagag attggtggtc tcatctcagt 6180

aagcatcttc attctttcag agaaatatgc agaatataat ggatcatcct tctgatgctc 6240

gttgatatac ataatccttg atctagggac ttccttcaag aaaaaacata tatcctatcc 6300

gccgatgagc ccattgtttg aaaggaactt atcccatcct tggactttga atggccgctc 6360

atcagcattc ttaaagagca gagtgtcaca tgttgcatcg ttgaactcca ccctaacgtt 6420

gcatgggata cactatgcaa caatagagaa aaatgcagta aataattatt ggtaaagata 6480

ctaaaataag tgcaattgtg tgctaaagtt aaagaatatt accacggatt attcgaattc 6540

ctcattcaag taaagaccgg atgacactcc agataacatg ctgtttgcgc ggtcagattt 6600

ctagatttgg cacatcatcc tgttacgttt tctttagagg aggatcatcc ccggcctctg 6660

catctgatcg atgcatgcat ccattttatt aattattcac caaagacctt acaaagaaat 6720

acaacaataa gtctcaagtc atcgtctatg cgacaactgt cgctactcct atccagctga 6780

tgaagggatg ctgatagtcc gggcctaata ccacagacct cgcagacaag cctaacatct 6840

aagacctgag gccccaacca agccacttgc cgggtatggg cacacaccgg tccggcgcgc 6900

tctcagatgc cgccgccgcc aactgccact actccatctt cagagctgta ctgacgcatc 6960

attcttgccc ggtctagctg tcgtcaacgc caccacgacg cccaatggca ccaccaccct 7020

gtgcgcaaac tgctgagcac gtcgcggtcg ccgccggtac acctcagcac catgccgcca 7080

agtaccatca gtcgacacag cttgaagtct ctgggagatc tgtcatgcgt agcacctgcc 7140

aaccatgcat gacaaagcgt agtagtgtta cgtgcatgac ttattttatc cctacgaaca 7200

ttttcccaaa atttattcac gtgtagctta aaacaacata atttctaagt atttgcctct 7260

atacaatcaa tatcaccaac acctatcaag caaaagtcaa acttgacgtt gcaagtgcac 7320

ccctgcagta aatatttgac atactagttt atcaaattaa tatttgagaa catctaagct 7380

cgcagcgaag aggccgagga cataaaaagg tcttcgccag aaaaaatcag catggttttt 7440

gctaaaaagt tgaacatatt gagtgccaaa aaagttgtcg aaagagaaat taatcacatt 7500

ttgcctgaac attcatgaat ccctgaattt ttgaaaatgg aagaattctg aatacctaca 7560

ctatttaatc caaaggggtc aattaagacc tacattgcta aacgaaccaa tacatatata 7620

tcagacacaa acttgatttt tatgtaatag aaaggtccat gcgaacctta aactcgactt 7680

gtaacagaaa cttcccagcg aggttttgac cctaaccctc tccaggactt gtgtgtcgta 7740

gacgacacat ccacaggtga ggaaattaaa cgcctggctt ctggattcta catggaaatg 7800

tgatcatcta tggtgatata cgaatgcgtg cagtgtgcgt gaacggggtg gtgtgcaagg 7860

aaccgaagac catggtggtg ggaaacgtct tctttgtcag cgaggacagg cccggcaacg 7920

cggacaaaaa ccgctatgtg ttcttggtgc tgaacatgca gagcccaggc ttgaacacgc 7980

tactcctggt ggcggcgcgg ttcatacatc tagcgagaga caggacgagc agcaagggga 8040

gagggacggg tggcttatgt accaacgatt aagtaggaac taaggcagat ccggcgacgg 8100

gcgcctgcgg tggagcgacg gtgcggtccg atggcgaagg atttggatgg gtccagggtt 8160

tggatttggg gggtaatgtt caggaatgac gaagggttat gcctgcacgc caagacttag 8220

aagtcgcagc ctcccatgcg acatcacagg ccctccagtt atgcatattc acagtgtcgg 8280

gcaattttag gtgcctgcca caactaatat tttgccaatt tctggtaata atcttaaatt 8340

ttttctttat tttaggacta tatttctgtt ctcctttctt tgtggggttt aaagtaactt 8400

cttagaaact cccatgcccg aatttgggac taatctattt tttctttgtt atttttaaag 8460

cattaaatga gtttctagcc actaaaatga gcaaaatgct cattgcttcg aaaaaagagg 8520

caaatgagca ctgaaaagga tgaaccttgg catgtgttca ttatatgact cctatagggt 8580

gtgctaaaaa tctgagaacg ttatgggcaa aacgtgatgc acttcattta caaactgaac 8640

actcttggat aaagtatcag gctttcggat ggaaaccctt gcctcccata gagaattctt 8700

tctccttcga acttttttac ttcatttccc cttgaacgtt ttcgatgtca aaacatacac 8760

aactgcaagt ctcatgctca aatttgagac tactctaggt tggtttgcta attttaaagc 8820

attaactgca tttctaacca cttaaatgag caaaattctc ttcgattcat aaccacatac 8880

acatgagtta taaaaatgat gaaacttgga atgggttcat tatatgattt ctacaaggtg 8940

tggtataaat tttagagcgt tacgggagaa acatgatgca ctttgtgtac aaactgaact 9000

ctctcaaatg aagtatcgga ttttcaaacg gaaacgttgg acttccacat agaacttaat 9060

tcttgtgcat catttccctt caaacttttt ctatatttaa tatacatagt taaaagtctc 9120

atgatcaaaa tggggactat tcttggttga tttgctattt ttaaagtatt aaatgatttt 9180

ctagctatta aaaggagcaa aatgctctta cattcaaaat aaggtgcaaa taagctttaa 9240

aaaggataaa tgtggcaagg attcattata tgacacatat aggcagtggt aaaattttga 9300

aagcgttacg ggataaactt aatgcacttc ttctacaaaa tgaacactct ccgaagaagc 9360

atcagggttc tgaacggaaa ccttggactt caacaaagaa ctcaattttg ttaaatcatt 9420

tcccttttaa acctttttat atgtttaata cacacagtta aaagtctcat gatcaaatgt 9480

ggtactattc tgggttgatt tgctattttt aaggcattaa gggccagtta tttttggcgg 9540

tttagaaaag taagttgact ctccccagct taaataataa gtcatcttct aaatttgtaa 9600

ggcttctaaa acaagttatc ccataactag tgtagaagcc ccaacgaata agagaggtgg 9660

ttatgaaaaa agctggggaa aaggtggctt attttctatg ttgccgtcaa ttattttcta 9720

cccattaaga ggttcagaat gccctttgat tcaaaataat atgaaatgag ctctaaaagc 9780

gatgaaactt ggcatggttc ataatatgac cccctacagg gtgtgcggac aaaaataatt 9840

aagagcgtta cgcgaaaaac ttgatgcact tcgtgtataa actgaacact ctgtgacgaa 9900

gtatcaggat ttcgaacgca aatctttgac ttactcatag aattcaattt ttttatatca 9960

tttccacttc aaactgtatg tttaatatac acaactgaaa ctcccatgct caaatttggg 10020

actattctgg gttaattaat atgatttttt ttcaaaaaat ttccttactt attagatcta 10080

cggtgggatc ctttttgtgc cagccgctgc tatcttttga attttttaga aaattttgac 10140

gtcgcttagc gaaatattgc taaggtgggc accttactgc ccgtcattgg tatttcacaa 10200

accaatgacg gaccgtagtt tgttgcccat cactgctgtt tttgtttcac gactaatttt 10260

gcaaaactgt acgagtggcg cgtgtccaat ttgcccacca ctgatgttcg tatagcatgg 10320

caggccacgt ttatcacccg ccactcgtac tataccaaaa tagcaatgac gggttccaga 10380

cctcaccttc cactaaagtg accccaccta tatataagcc ttttaaccag tagtgtatct 10440

ggctctcaca cacacatact ctaggttcct cttaacacaa aatctctctt cctccctaca 10500

cacagtctat ccatctctct ctagatgtat gcccacatag gtgggtgact gccgtcgtcg 10560

acgcggatag ggtttgggat tgacatctca cgggagagag acaaggggga caagatattt 10620

acaccactgg ccgggaatcc actagtttta tttgagagtt gagactattc gatgacgagt 10680

gtgtccagtg ggctttctgg cagtgcctgc ttttctctct ttcccgtccc atgtataact 10740

ctattctgag ttgtattttt gttatcagta acgggaaggg tatacccggt taaaagaaaa 10800

ggctgtattt ggtgaggtat atgtttgtat agtgggaaaa tgataatata tgggtagcgg 10860

ccggacgcat cttgagttct tgacgacaga ttccttacca gtacctacta ccgttagcgg 10920

taaaaatgcc tttgcgagta gctaccacgt taacacttgg cccagtcttt cctttcgtcg 10980

tcgtcagtcc acgtaaaaga aatgctattg aaaattgaca aagtgttatc cctgtatttc 11040

caaggtgttg gaggacccga gcatggtgaa gcggatcagc acgtctttct gggagctgca 11100

cttgccgtca tccttggagt cgaaggatcc gagctccagc acatcagcaa acgataccag 11160

ggaggccacc gacatcatct tgttcgagga tttcgaccac aacgacacag ttgagggggt 11220

gatctctgag caaagggagg tccagtgccc gtccaacgtc aatctggagc gcctcacaaa 11280

gcagatggac gagttccaca gccttctcgg tggactggac gtgcatcctc tcgaagacag 11340

atggatcatg gacgagccct ttgagtttac gttttcccca gaagtggcgc cggctatgga 11400

tatgccgagc accgacgatg tcatcgtcac tttaagtagg tccgaaggct ctcgtccatc 11460

ctgcttcaca gcgtggaagg gatcatccga gtcgaaatac gtggctggcc aggtcgttgg 11520

ggagtcacag aagttgctga ataaagttgt ggctggtggt gcatgggcga gcaattatgg 11580

cggtcgcacc atggtgagag ctcagggaat taacagcaac acccatgtca tgacagagag 11640

aagacgccgg gagaaactca acgagatgtt cctggttctc aagtcactgg tcccgtccat 11700

tcacaaggta gtaatatgta aagtccatgt acctttagca aactgcacac gttgtttttc 11760

atagcttttt tactgttgga ttgcaggtag acaaagcatc catcctcaca gaaacgatag 11820

gttatcttag agaactgaag caaagggtag atcagctaga atccagccgg tcaccgtctc 11880

acccaaaaga aacaacagga ccgagcagaa gccatgtcgt cggcgctagg aagaagatag 11940

tctcggccgg atccaagagg aaggcgccag ggctggagag cccgagcaat gtcgtgaacg 12000

tgacgatgct ggacaaggtg gtgctgttgg aggtgcagtg cccgtggaag gagctgctga 12060

tgacacaagt gtttgacgcc atcaagagcc tctgtctgga cgttgtctcc gtgcaggcat 12120

ccacatcagg tggccgtctt gacctcaaga tacgagctaa tcagcaggta tatatagatc 12180

gcactaattt ctacgaccga tctggcagta tataaggaaa tgtatagcct gacttaagga 12240

tccatgatta ccgtctcatt cactgacatt gctggattgt tgcagcttgc ggtcggttct 12300

gctatggtgg cacctggggc aatcaccgaa acacttcaga aagctatata g 12351

<210> 8

<211> 1683

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 8

atggcgctat cagctcctcc cagtcaggaa cagccgtcgg ggaagcaatt cggctaccag 60

ctcgctgctg ctgtgaggag catcaactgg acttatggca tattttggtc catttccgcc 120

agcccgcgcc caggccactc ctcagttctg gcgtggaagg atgggttcta caacggcgag 180

ataaagacta gaaagattac cggctcgacc actacggagc ttacagcgga cgagcgcgtc 240

atgcacagaa gcaagcaact gagggagctc tacgaatcgc tcttgcccgg caactccaac 300

aaccgggcaa ggcgaccaac cgcctcactg tcaccggagg atctcgggga cggcgagtgg 360

tattacacca taagcatgac ttacaccttc caccctaatc aagggttgcc aggcaaaagc 420

tttgcgagca atcaacatgt ttggctgtac aacgctcaat acgcaaacac cagagttttc 480

ccccgcgcgc tcttagcaaa gactgcttct attcagacaa tcgtttgcat tcccttcatg 540

ggcggtgtgc ttgagctcgg aacgtcggat caggtgttgg aggacccgag catggtgaag 600

cggatcagca cgtctttctg ggagctgcac ttgccgtcat ccttggagtc gaaggatccg 660

agctccagca catcagcaaa cgataccagg gaggccaccg acatcatctt gttcgaggat 720

ttcgaccaca acgacacagt tgagggggtg atctctgagc aaagggaggt ccagtgcccg 780

tccaacgtca atctggagcg cctcacaaag cagatggacg agttccacag ccttctcggt 840

ggactggacg tgcatcctct cgaagacaga tggatcatgg acgagccctt tgagtttacg 900

ttttccccag aagtggcgcc ggctatggat atgccgagca ccgacgatgt catcgtcact 960

ttaagtaggt ccgaaggctc tcgtccatcc tgcttcacag cgtggaaggg atcatccgag 1020

tcgaaatacg tggctggcca ggtcgttggg gagtcacaga agttgctgaa taaagttgtg 1080

gctggtggtg catgggcgag caattatggc ggtcgcacca tggtgagagc tcagggaatt 1140

aacagcaaca cccatgtcat gacagagaga agacgccggg agaaactcaa cgagatgttc 1200

ctggttctca agtcactggt cccgtccatt cacaaggtag acaaagcatc catcctcaca 1260

gaaacgatag gttatcttag agaactgaag caaagggtag atcagctaga atccagccgg 1320

tcaccgtctc acccaaaaga aacaacagga ccgagcagaa gccatgtcgt cggcgctagg 1380

aagaagatag tctcggccgg atccaagagg aaggcgccag ggctggagag cccgagcaat 1440

gtcgtgaacg tgacgatgct ggacaaggtg gtgctgttgg aggtgcagtg cccgtggaag 1500

gagctgctga tgacacaagt gtttgacgcc atcaagagcc tctgtctgga cgttgtctcc 1560

gtgcaggcat ccacatcagg tggccgtctt gacctcaaga tacgagctaa tcagcagctt 1620

gcggtcggtt ctgctatggt ggcacctggg gcaatcaccg aaacacttca gaaagctata 1680

tag 1683

<210> 9

<211> 560

<212> PRT

<213> Elytrigia elongata (Elytrigia elongata)

<400> 9

Met Ala Leu Ser Ala Pro Pro Ser Gln Glu Gln Pro Ser Gly Lys Gln

1 5 10 15

Phe Gly Tyr Gln Leu Ala Ala Ala Val Arg Ser Ile Asn Trp Thr Tyr

20 25 30

Gly Ile Phe Trp Ser Ile Ser Ala Ser Pro Arg Pro Gly His Ser Ser

35 40 45

Val Leu Ala Trp Lys Asp Gly Phe Tyr Asn Gly Glu Ile Lys Thr Arg

50 55 60

Lys Ile Thr Gly Ser Thr Thr Thr Glu Leu Thr Ala Asp Glu Arg Val

65 70 75 80

Met His Arg Ser Lys Gln Leu Arg Glu Leu Tyr Glu Ser Leu Leu Pro

85 90 95

Gly Asn Ser Asn Asn Arg Ala Arg Arg Pro Thr Ala Ser Leu Ser Pro

100 105 110

Glu Asp Leu Gly Asp Gly Glu Trp Tyr Tyr Thr Ile Ser Met Thr Tyr

115 120 125

Thr Phe His Pro Asn Gln Gly Leu Pro Gly Lys Ser Phe Ala Ser Asn

130 135 140

Gln His Val Trp Leu Tyr Asn Ala Gln Tyr Ala Asn Thr Arg Val Phe

145 150 155 160

Pro Arg Ala Leu Leu Ala Lys Thr Ala Ser Ile Gln Thr Ile Val Cys

165 170 175

Ile Pro Phe Met Gly Gly Val Leu Glu Leu Gly Thr Ser Asp Gln Val

180 185 190

Leu Glu Asp Pro Ser Met Val Lys Arg Ile Ser Thr Ser Phe Trp Glu

195 200 205

Leu His Leu Pro Ser Ser Leu Glu Ser Lys Asp Pro Ser Ser Ser Thr

210 215 220

Ser Ala Asn Asp Thr Arg Glu Ala Thr Asp Ile Ile Leu Phe Glu Asp

225 230 235 240

Phe Asp His Asn Asp Thr Val Glu Gly Val Ile Ser Glu Gln Arg Glu

245 250 255

Val Gln Cys Pro Ser Asn Val Asn Leu Glu Arg Leu Thr Lys Gln Met

260 265 270

Asp Glu Phe His Ser Leu Leu Gly Gly Leu Asp Val His Pro Leu Glu

275 280 285

Asp Arg Trp Ile Met Asp Glu Pro Phe Glu Phe Thr Phe Ser Pro Glu

290 295 300

Val Ala Pro Ala Met Asp Met Pro Ser Thr Asp Asp Val Ile Val Thr

305 310 315 320

Leu Ser Arg Ser Glu Gly Ser Arg Pro Ser Cys Phe Thr Ala Trp Lys

325 330 335

Gly Ser Ser Glu Ser Lys Tyr Val Ala Gly Gln Val Val Gly Glu Ser

340 345 350

Gln Lys Leu Leu Asn Lys Val Val Ala Gly Gly Ala Trp Ala Ser Asn

355 360 365

Tyr Gly Gly Arg Thr Met Val Arg Ala Gln Gly Ile Asn Ser Asn Thr

370 375 380

His Val Met Thr Glu Arg Arg Arg Arg Glu Lys Leu Asn Glu Met Phe

385 390 395 400

Leu Val Leu Lys Ser Leu Val Pro Ser Ile His Lys Val Asp Lys Ala

405 410 415

Ser Ile Leu Thr Glu Thr Ile Gly Tyr Leu Arg Glu Leu Lys Gln Arg

420 425 430

Val Asp Gln Leu Glu Ser Ser Arg Ser Pro Ser His Pro Lys Glu Thr

435 440 445

Thr Gly Pro Ser Arg Ser His Val Val Gly Ala Arg Lys Lys Ile Val

450 455 460

Ser Ala Gly Ser Lys Arg Lys Ala Pro Gly Leu Glu Ser Pro Ser Asn

465 470 475 480

Val Val Asn Val Thr Met Leu Asp Lys Val Val Leu Leu Glu Val Gln

485 490 495

Cys Pro Trp Lys Glu Leu Leu Met Thr Gln Val Phe Asp Ala Ile Lys

500 505 510

Ser Leu Cys Leu Asp Val Val Ser Val Gln Ala Ser Thr Ser Gly Gly

515 520 525

Arg Leu Asp Leu Lys Ile Arg Ala Asn Gln Gln Leu Ala Val Gly Ser

530 535 540

Ala Met Val Ala Pro Gly Ala Ile Thr Glu Thr Leu Gln Lys Ala Ile

545 550 555 560

<210> 10

<211> 4003

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 10

atggcgctat cagctcctcc cagtcaggaa cagccgtcgg ggaagcaatt cggctaccag 60

ctcgctgctg ctgtgaggag catcaactgg acgtatgcca tattttggtc catttccgcc 120

agcccgcgcc caggccactc ctcgtaatga ataaatgacc actctctatc tctatatgta 180

tcaatagata tcatatttgg gtttgtagca aacccacaaa gttcgtactt ttcatatata 240

tagtgtccgg cgtcgacgcc gtcggttcgt ttcgcagagt tctggcgtgg aaggatgggt 300

tctacaacgg cgagataaag acaagaaaga ttaccggctc gaccactacg gagcttacag 360

cggacgagcg cgtcatgcac agaagcaagc aactgaggga gctctacgaa tcgctcttgc 420

ccggcaactc caacaatcgg gcaaggcgac ctgccgcctc actgtcaccg gaggatctag 480

gggacggcga gtggtattac accataagca tgacttacac cttccaccct aatcaagggt 540

acgtagttat caaccatgat gtatgcgata tatgtgcctg ccatgattca tgccgtccat 600

atttttatat acgggtacat gacaaagaca aattttgcgg ttatgaaaat agtcacaaac 660

atgcactatt ttttgttaca aattacatat taatttgtac cttttttagt agttatcttg 720

ttaatatata ctggcatgtt gttgccagga aaagatcact tcaagaaaat tataaacaag 780

catatataat acgtccatac atatgtccaa ttattttcag ccttttcctt tttctaaatt 840

aaatacatag agtccaagct tcaaatcctg cacttaaacc tcaagctaat ctgttacact 900

cccttgcagc tagtctctta ctagtattat ctgttctttt tttgaaggaa aaactttcga 960

tctattcgtc atcatcacag taaaaaaaac actagaagta acataaatta cattcatgtc 1020

tgtagaacac cttgcgacga ttgcaaacat tggtgtgcat gccgaaggcg cgctacactc 1080

atcgcccctc cctcattgga gcttggcaaa cttaattgta gtagacggta gggaagtctt 1140

tatgctataa ggccttaaag gaccagcgca ccagaacaac aaccgtgacc attaaagaaa 1200

agcaaaaaga aaagaaaagc atagatggaa aagattcaac atgtagacat ataacacagc 1260

aaaactaagg ccgaatccaa gtatatccac taaactcaaa cactgactga atcccgcgag 1320

attcaccgac aatgccctcc gacaatgtta gatgcatcat tgggataggg acttggcagg 1380

gtgaacctta ttctgtcttc agatagcaga tgtcgcctca ccttcctaag cagcacacaa 1440

acactaaata aaacttgaaa aacacttaaa atgaagtagg agcactctca ctggcaagag 1500

ccacgatcca ttatgcctcc atggccctaa agccggagga agatcgatgg cagcatcggg 1560

gggaggcgag gaagccctcg tttcctggga gttcgcttgt ggagcactaa tactatcttg 1620

atactagcta ccatagactg cagtggtaag atgtacatac tgaagtgatt tttttttctg 1680

tcggcgtgaa tgtacttact accaggttgc caggcaaaag ctttgcgagc aatcaacatg 1740

tttggctgta caacgctcag aacgcaaaca ccagagtttt cccccgcgcg ctcttagcaa 1800

aggtatatct cttctactta tctcatatta tgtgtgtata gcaatgatgc gggtcgggtt 1860

ttcaaccccc ttacataaga aaacatcgtt tggtctttaa ctaataataa tactccaaaa 1920

cgtgcatgat gtcttcctac accatccgat ggtcaaatca gaccgcttct attcaggtca 1980

gcacattact tcctactata gatagataga tagacgcaca catatacaca tctctgatct 2040

caaaccgttc acatatttcc agacaatcgt ttgcattccc ttcatgggcg gtgtgctgga 2100

gctcggaacg tcggatcagg tcggtgcaca tgagtttcaa tttatgttct cagtaactct 2160

cagcatgttc ttccgacttg ccgacgggtt caacggtaac cttttaatat taggggtagc 2220

tattttgttt gccaatacat gctagctatt tttgtatgtg ttgtcccagt ctaccattct 2280

gccccttttg catttatgcg ttattccttt ttagtacatt aatttacact agtcttccac 2340

tgcgttttcc tactactatg tattttatat tatatttgtg attatcttta gagcaagtac 2400

aatagagtga tgtaggcggg ctacaagaga tggcacatca cttttatgcc tagttgaagg 2460

agagaggaga ggagagaaga gaagcgggtt acaaacttac agcaaaagtg accccaccta 2520

tatataaggc ttttaatcag tagtgtctgc ctcacacaca cagatactct aggttcctct 2580

taacacacca tctccctctg ttcgactgtc cctacacaca gtctctatcc atctctctct 2640

agatgtatgc ccccatagga gggtgactgc cgtcgacgtg gatagtgatt gggattgaga 2700

tctcatggca agggggacca gatatttaca ccactggcga gggatccact agttgtattt 2760

gagagttgag actatcgatg acggatgcgt tcaatgggtt tttggcagtg cgtgaacttg 2820

cttttctctc tttcccgtcc cccgtataac tctattctga gttgtattat tgttatcagt 2880

aacgaaaagg gtatacccga ttaaaagaaa aggctgtatt tgaggtactc atgtatatat 2940

gttttgtata gtgggaaaaa tgataatata tgtgtagcgg ccgcacgcat cttgaggaca 3000

gattccttac cagtacgtac caccatagcg gtaaaaatgc ctttgcgagt agctaccaca 3060

ttaacacttg gccaagtttt tcctttcgtc gtcgtccgtc cacgtaaagc aaatgctatt 3120

gacgattgac aaagtgtatc ctatatttcc aaggttttgg aggaccccgg catggtgaag 3180

cggatcagca cgtctttctg ggagctgcac ttgccgtcat ccttggagtc gaaggatgcg 3240

agctccagca catcagcgaa cgataccagg gaggccaccg acatcatctt gttcgaggac 3300

ttcgaccaca acgacacagt tgagggggtc acctctgagc aaagggaggt ccagtgcctg 3360

tccaacgtca atctggagcg cctcacaaag cagatggacg agttccacag ccttctcggt 3420

ggactggacg tgcatcctct cgaagacaga tggatcatgg acgagccctt tgagtttacg 3480

ttttccccgg aactggcgtc ggctatggat atgccgagca ccgacgatgt catcgtcact 3540

ttaagtaggt ctgaaggctc tcgtccatcc tgcttcacgg cgtggaaggg atcatccgag 3600

ttgaaatacg tgcctggcca ggtcgttggg gagtcacaga agttgctgac taaagttgtg 3660

gctggtggtg catgggcgag caattatggc ggtcgcacca cggtgagagc tcaggaaatt 3720

aacaacaaca cccatgtcat gacagagaga agacgccggg agaaactcaa cgagatgttc 3780

ctggttctca agtcactagt cccgtccatt cacaaggtag taatatgtag agtccattta 3840

ccatcagcaa attgcacacg ttgtttttca tagttttttt actgctggat tgcaggtgga 3900

caaagcatcc atcctcacag aaacgatagg ttatcttaga gaactgaagc aacgggtaga 3960

tcagctagaa tccagccggt caccgtcttg ttggatatta tga 4003

<210> 11

<211> 1344

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 11

atggcgctat cagctcctcc cagtcaggaa cagccgtcgg ggaagcaatt cggctaccag 60

ctcgctgctg ctgtgaggag catcaactgg acgtatgcca tattttggtc catttccgcc 120

agcccgcgcc caggccactc ctcagttctg gcgtggaagg atgggttcta caacggcgag 180

ataaagacaa gaaagattac cggctcgacc actacggagc ttacagcgga cgagcgcgtc 240

atgcacagaa gcaagcaact gagggagctc tacgaatcgc tcttgcccgg caactccaac 300

aatcgggcaa ggcgacctgc cgcctcactg tcaccggagg atctagggga cggcgagtgg 360

tattacacca taagcatgac ttacaccttc caccctaatc aagggttgcc aggcaaaagc 420

tttgcgagca atcaacatgt ttggctgtac aacgctcaga acgcaaacac cagagttttc 480

ccccgcgcgc tcttagcaaa gaccgcttct attcagacaa tcgtttgcat tcccttcatg 540

ggcggtgtgc tggagctcgg aacgtcggat caggttttgg aggaccccgg catggtgaag 600

cggatcagca cgtctttctg ggagctgcac ttgccgtcat ccttggagtc gaaggatgcg 660

agctccagca catcagcgaa cgataccagg gaggccaccg acatcatctt gttcgaggac 720

ttcgaccaca acgacacagt tgagggggtc acctctgagc aaagggaggt ccagtgcctg 780

tccaacgtca atctggagcg cctcacaaag cagatggacg agttccacag ccttctcggt 840

ggactggacg tgcatcctct cgaagacaga tggatcatgg acgagccctt tgagtttacg 900

ttttccccgg aactggcgtc ggctatggat atgccgagca ccgacgatgt catcgtcact 960

ttaagtaggt ctgaaggctc tcgtccatcc tgcttcacgg cgtggaaggg atcatccgag 1020

ttgaaatacg tgcctggcca ggtcgttggg gagtcacaga agttgctgac taaagttgtg 1080

gctggtggtg catgggcgag caattatggc ggtcgcacca cggtgagagc tcaggaaatt 1140

aacaacaaca cccatgtcat gacagagaga agacgccggg agaaactcaa cgagatgttc 1200

ctggttctca agtcactagt cccgtccatt cacaaggtgg acaaagcatc catcctcaca 1260

gaaacgatag gttatcttag agaactgaag caacgggtag atcagctaga atccagccgg 1320

tcaccgtctt gttggatatt atga 1344

<210> 12

<211> 447

<212> PRT

<213> Elytrigia elongata (Elytrigia elongata)

<400> 12

Met Ala Leu Ser Ala Pro Pro Ser Gln Glu Gln Pro Ser Gly Lys Gln

1 5 10 15

Phe Gly Tyr Gln Leu Ala Ala Ala Val Arg Ser Ile Asn Trp Thr Tyr

20 25 30

Ala Ile Phe Trp Ser Ile Ser Ala Ser Pro Arg Pro Gly His Ser Ser

35 40 45

Val Leu Ala Trp Lys Asp Gly Phe Tyr Asn Gly Glu Ile Lys Thr Arg

50 55 60

Lys Ile Thr Gly Ser Thr Thr Thr Glu Leu Thr Ala Asp Glu Arg Val

65 70 75 80

Met His Arg Ser Lys Gln Leu Arg Glu Leu Tyr Glu Ser Leu Leu Pro

85 90 95

Gly Asn Ser Asn Asn Arg Ala Arg Arg Pro Ala Ala Ser Leu Ser Pro

100 105 110

Glu Asp Leu Gly Asp Gly Glu Trp Tyr Tyr Thr Ile Ser Met Thr Tyr

115 120 125

Thr Phe His Pro Asn Gln Gly Leu Pro Gly Lys Ser Phe Ala Ser Asn

130 135 140

Gln His Val Trp Leu Tyr Asn Ala Gln Asn Ala Asn Thr Arg Val Phe

145 150 155 160

Pro Arg Ala Leu Leu Ala Lys Thr Ala Ser Ile Gln Thr Ile Val Cys

165 170 175

Ile Pro Phe Met Gly Gly Val Leu Glu Leu Gly Thr Ser Asp Gln Val

180 185 190

Leu Glu Asp Pro Gly Met Val Lys Arg Ile Ser Thr Ser Phe Trp Glu

195 200 205

Leu His Leu Pro Ser Ser Leu Glu Ser Lys Asp Ala Ser Ser Ser Thr

210 215 220

Ser Ala Asn Asp Thr Arg Glu Ala Thr Asp Ile Ile Leu Phe Glu Asp

225 230 235 240

Phe Asp His Asn Asp Thr Val Glu Gly Val Thr Ser Glu Gln Arg Glu

245 250 255

Val Gln Cys Leu Ser Asn Val Asn Leu Glu Arg Leu Thr Lys Gln Met

260 265 270

Asp Glu Phe His Ser Leu Leu Gly Gly Leu Asp Val His Pro Leu Glu

275 280 285

Asp Arg Trp Ile Met Asp Glu Pro Phe Glu Phe Thr Phe Ser Pro Glu

290 295 300

Leu Ala Ser Ala Met Asp Met Pro Ser Thr Asp Asp Val Ile Val Thr

305 310 315 320

Leu Ser Arg Ser Glu Gly Ser Arg Pro Ser Cys Phe Thr Ala Trp Lys

325 330 335

Gly Ser Ser Glu Leu Lys Tyr Val Pro Gly Gln Val Val Gly Glu Ser

340 345 350

Gln Lys Leu Leu Thr Lys Val Val Ala Gly Gly Ala Trp Ala Ser Asn

355 360 365

Tyr Gly Gly Arg Thr Thr Val Arg Ala Gln Glu Ile Asn Asn Asn Thr

370 375 380

His Val Met Thr Glu Arg Arg Arg Arg Glu Lys Leu Asn Glu Met Phe

385 390 395 400

Leu Val Leu Lys Ser Leu Val Pro Ser Ile His Lys Val Asp Lys Ala

405 410 415

Ser Ile Leu Thr Glu Thr Ile Gly Tyr Leu Arg Glu Leu Lys Gln Arg

420 425 430

Val Asp Gln Leu Glu Ser Ser Arg Ser Pro Ser Cys Trp Ile Leu

435 440 445

<210> 13

<211> 1952

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 13

ctccgctatg tctatggcac tatggatctt ggcgtcacgc ttcacgcctc cgccgacacc 60

gccctcaccg cctactccga tgcagactgg gcgggctacc ctgacactca tcgctccacc 120

tcgggctatt gtgtctacct tggaccctca cttatttcgt ggtcgtccaa gcggcagcct 180

acggtctctc gttccagtgc tgaggctgag tatcgtgcgg tggccaacgc cgtcgccgag 240

tgttcgtggc ttcgccagct gcttcaggag ctttcctgcc ctgttgactg tgccacggtg 300

gtctactgcg acaacgtctc ggcggtctac ctctccgcta acccggtgca tcatcgacgg 360

accaagcata ttgagttgga tattcatttt gttcgggaac aggtggccct tggtcatatt 420

cgtgttttac accaggggcg gaactgggcc tggggcaact ggggctatag ccccaggcat 480

ggcccatcta gtaggctacc cagcaggaat tttcccatat attagtacac ttgtagaggc 540

ccagccccag gcctcagccc acagagctct ccagtttgga cgatcgctcc aaagaatcac 600

cagcagccac gatgcaagag cgctccgatc ctcacgcttc gtctcggtag atcgaccgaa 660

aagtctcacg ctgcgccgcc aactctctcg actgctcgac tccagcagac gctcgcccag 720

gcggccggcg ctcggaaccg cggatctatc gcgccgtttg aataaagcaa tctccatgcc 780

tgatctcctg tgatccacac ccgattgaaa aggtaatgct aaatacctag gtttgtcgta 840

tcatctaata tttcccccta gttccttttt atcagctgaa atcaatgaat tggtgatgtt 900

aaattgtatt gctttagtca attttattcc atcgttcttt gttaacaggg gaaaaataga 960

aatccaagat gaagtggacc cggacagttt ttcgtgatca tggtataata ttttcttgct 1020

tcccaaaatt tcatatgaac aataacttgt ctatgtctag ttcttgattt taaatactca 1080

actttatgtt tacagctccg ggtcctagct actattgctt gtgaagaaac gatggcctca 1140

agtgagacaa gccttcatta atttggtacg actgtttgta atgatgcact ttgttatatt 1200

gatgagatat ttattaaatt tgctcttatg gtaataagta attcatattc tattcttaat 1260

ctgaatgtgt ggtgtacttc taacatgcat tggtacgtag agtatttggc attttatttc 1320

ctttaggttg tgatatttgg cgttatttgt agtggtttag ctttagtcct aggcttcgag 1380

aaatcctggc tccgcctctg ttttacacgt tcctactttt caacaatttg cagatattat 1440

gaccaaaggc ttgcctacgg cgtcatttaa ggagttccgg tccagtcttt gcgtcagccg 1500

cggtgccgct tcgactgcgg ggggtgttga gtacatgtgt tatgtgtata ttgtgtattg 1560

ggtccgtctc ctagttcttt gtatagttga ggtctatggc ccaccgttgt acatcatata 1620

tacgtgccta tgcacgagag caatacatca tgcaatcata gtctcataca ctagcgatct 1680

gaagctttac accaggttat aggttttctt cctcgacttt ggacggatat catcatcggt 1740

gccgccggtg agatgtatag gcggcggaac ggcggtgctg aacgtccgtg ttcattgcac 1800

agatggatcc tttgctagcc gtttggtgta cctaactcgg acttaggtgt tgcttgcggt 1860

gaccacatgg tgtattaata aagcacaacg gccttctgcc cacggatcaa acacagtaca 1920

cacgggcagt tgggaaaaag ataacgggga gg 1952

<210> 14

<211> 2013

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 14

ggtcaccaaa acacataact cataatacaa atcatcatcg aacgctaagc gtgtggaccc 60

tacgggttcg agaactatgt agacatgacc gagacagatc tccggtcaat aaccaatagc 120

ggaacctgga tgctcatatt ggctcctaca tattctacga agatctttat cggtcaagcc 180

gcataacaac atacgtcatt ccctttgtca tcggtatgtt acttgcccga gattcgatcg 240

tcggtgtcat catacctagt tcaatctcgt taccggcaag tctctttact cgttccgtaa 300

tgcatcatcc cgcaactaac tcattagtca cattgcttgc aaggcttata gtgatgtgca 360

ttaccgagag ggcccagaga tacctctccg atacacggag tgacaaatcc taatctcgat 420

ctatgccaac tcaacaaaca ccatcggaga cacctgtaga gcatctttat aatcacccag 480

ttacgttgtg atgtttgata gcacacaaag tgttcctttg gtattcggga gttgcataat 540

ctcatagtca gaggaatatg tataagtcat gaagaaagca atagcaataa aactaaacga 600

tcataatgct aagctaatgg atgggtcttg tccatcacat cattctctaa tgatgtgatc 660

ccgttcttca aatgacaaca catgtctatg gtcaggaaac ttaaccatct ttgattaacg 720

agctagttaa gtagaggcat actagggcca ctctatttgt ctatgtattg acacatgtac 780

taagtttccg gttaatacaa ttctagcatg aataataaac atttatcatg atataaggaa 840

atataaataa caactttatt attgtctcta gggcatattt ccttcagtat gaaggccgac 900

tgatctgggt gggtgatgcg gttggccaaa ggagtcaccc tattgacgta cccctttgcc 960

aagatgcggg agatcacatt gatcacggta atcaccctgt tgcttttgta aagaatggag 1020

ttatgagaat aggtgaaata tgctttgtgg gacgaaattg actacgcatt ttgtgtataa 1080

tcagggttta gaagtccatt gtgtgtgcat gtttgatacg agatatgttg ctcgtatgaa 1140

aagaactata tctgcaaagc tatatttagt tcaaattaag atttgagcta cattattgca 1200

ctcgttcatt ttattgagat aattatggcc taactaggat gactgagaat tttttctatg 1260

attatacgtg caacgcacgt gcatacttac tagtaaaaat gaaaatctaa cgcaataagt 1320

gaaaaacggg gcggcatagt gcaacacttt gctctacagg gccaacatcc tcgttgaaca 1380

tgcactacaa atactccctc cgtaaagaaa tataagagcg ttagcgatcc tcttatattt 1440

ttattacgga gggagtacta gctaggaata tcgcaaagca gtcgtcgtct ctggctccgg 1500

tcaaaaccag agcaagactt tcgtctcgtc tttctctcgg gcgcccactg cacggcagga 1560

gcgtatctgg gactgggaca gggaggccaa ccacgcgtag tagtagcgga gcaccagctc 1620

atggcccgca atgcatgcaa gatgtaaccc acagtcccgg tcctgcgccc gacaggctac 1680

gcctggagga gtcgccactg ccatctgtcc acgccaggtg tgagagatcg gagtgggcct 1740

agcgatctga agttttacac caggttttct tcctcgactt tggacggata tcatcatcgg 1800

tgccgccagt gagatgtata ggcggcggaa cggcggtgct gaacgtccgt gttcattgca 1860

cagatggatc ctttgctagc cgtttggtgt acctaactcg gacttaggtg ttgcttgcgg 1920

tgaccacatg gtgtattaat aaagcacaac ggccttctgc ccacggatca aacacagtac 1980

acacgggcag ttgggaaaat gataatgggg agg 2013

<210> 15

<211> 2084

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 15

tttcttaggg cattatgatg atgacttggc atcactagtg cggagttact gtagcatgat 60

tctcggggtg tcacaacagt acatcctaga gatcttggat cgcgccaata tgaccaactg 120

taaccccact gctactcctg tcgacaccaa atccaaacta tccgctactg acagtccacc 180

agttcccgat cctactctat gtcgtagcat tgctggagcc ttccagtgcc tcacccgcac 240

tcaccccgat atctcctatg cggtacagca ggcctgccta tttatgcacg atcctcgcgc 300

ccccaccttc agttcgtcaa aaggatctta cgctatctca aggccacatc acattatgga 360

ctgcagctta ccacttctcc tggacatgat ctcgtggctt attccgacgc agattgggcc 420

ggctgtcccg ctacactgaa gtccacctct ggtttctgtg tgttccttgg ccccaacttg 480

gtatcttggt cctccaaacg ccaacacacc gtctcttgat cgagcgccga agctgagtat 540

agggcggttg caaattgcgt cgctgaatcc tgctggcttc gccagcttct acaggagcta 600

catcaacctt cgtctagcgc cacaatcgtt tacactgaca acatcagtgg gacgtacctc 660

tcctccaacc ccgtctagca ccaacgcacc aagcacgtca agatcgactt gcattttgtc 720

cgagatcggg tggcacttgg agaggctcgg atcctgcacg tttcgtccaa ctcccaattt 780

gcggacgtgt tcaccaaggg cctgcctaca tccatcttcc atgagtcttg ttccagtctg 840

aacatccacc gtgtggagct ccgactgcgg ggtgctgtta aagtgtgttg taatattgta 900

catagacttg tcacgttgag gcctgcatgc ccatctgttg aaactacctc cacctctctg 960

ggatcacgat cccccttctc aatttttttt ttgaacacag tacagacgca agcactcata 1020

tacacgcgca tacactcacc cctatgaacg cacacacgca caccctaccc ctatgagcac 1080

ccccgaaaga ctgagccagc atatcatctt gaaatttacg aaatcaccgt agtcacctcg 1140

tcgtcgacgg gaacgtctcc tcccactgaa tgcacatcgc cggaaatcct gaaatgaatc 1200

caggaataaa tgcgagcacc aggatttgaa ccctggtggg ttggggatac cacagtccct 1260

ctaaccatcc aaccacaggt tggttcgcat cccccttctc agttgatggc ctgcgtgcct 1320

acttcttcgg cagtagttgc agctctcaat atatgtactc tgactcccct aaaatatata 1380

tatatatact ctgaccactg gttgtaatgt aacacgaaca cacagtgagg aattccaaac 1440

cataacttgg ttgtggggtg accattccac agcgttttat gcaacgtggc ggcagactag 1500

gttattcaca caaaaaagat ccatgcaagt ttccacgtgc tgctgcttcc aacgttgaag 1560

tgttttctgt gtcgtgcgtc gatcgtattt ggatcttctc agcaacgata gcggcgagat 1620

ttcgttgtat ctatgtcttc agagcggttc gagtagattt tttccttcat ttttcggact 1680

gaagaaaaag aggaaatgct accttccatc gttcgtactg tgtagttgtt ggtgcaacag 1740

cccgcgcatt tgcataaggt gccttgtata tatgtggaaa cgttcagttg ctacgtacct 1800

cttcgtggtc tgcacacgca tacagtctgc acactgatac cttaaagtaa gctttaattt 1860

tgcttcagtt tacttctcca gttcttggct agctgaatct cgacttctag tttggtcgga 1920

atttatataa ctttcctgcg tgtacagtac acagcgtatg taccatgcat ataactttat 1980

atacctttct tgatttcttt tgctgcttgt ttcctcgacc tagcgtctca ccttgtgagt 2040

tgtgcatgcg ggaaatagct actcagcggt gtggtaatcg atca 2084

<210> 16

<211> 2022

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 16

caacccaagc atctcttttc tgtcaccacc accatggaca tttctcccat accttctacc 60

tataaatctg ccttaaaaaa tccccattgg tacaatgtaa tgcttgaaga atttaatgct 120

ttgtagaatg atacatggtg tttggttcct cgacctgcag gtgtcaacat ggtcaccgag 180

aaatggatct ttaggcacaa atttcattcg gacggctccc tagctcgcta caaggcccat 240

tgggttctcc gtggattcac tcagcaggag ggcgtcgact acactgagat attttgtccg 300

gttataaaac cggccacagt tcgggtgctt ctcagcctcg ccaccactca ttcttggccc 360

attcatcaaa tggatctcaa gaacgccttc ctccatggca ctctcggtga aaccgtttac 420

tgcaatcaac cggctgggtt cattgattca tctcaccgca cccatgtttg tcttctcaag 480

aaatctttat attggttaaa acaggctcct cgcacatggt ttatctgttt ccaggccttc 540

atcctttcct tgggatttgt tgcttctaag tgcgactctt cactgttcat tcttcatcgt 600

ggctcggcca ttgcctatct tctcctttat gttgatgata ttatcctcac tgttaacacc 660

accgccacct tacactccat catcttctcg ctcaagcccg agttatctat gtttgacttc 720

ggtgacatac atcatttcct tggtgttaat gttacccgtt ccccacgtgg cctcttttta 780

tctcaagaat agtacatctt agagatcttg gattgcgcca atatgaccaa ctgtaacccc 840

attgctactc ctgtcaacac caaatccaaa ctatccgcta ctgacagtcc accagttccc 900

gatcctactc tataccgtag cattactgga gccctccagt acctcaccct cactcgcccc 960

gatatctcct atgcgttaca gcaggcctgc ctatttatgc acgatcctag cgcctcccac 1020

cttcagtttg tcaaaaggat cttacgctat ctcaaggcca catcagatta tggactgcag 1080

cttaccactt ctcctggaca tgatctcatg gcttattccg atgcagattg ggccggctgt 1140

cccgctacac tgaagtccac ctccggtttc tgtgtgttcc ttgaacccaa cttggtctct 1200

tggtcctccg agcggcaaca cactgtctct tgatcgagcg ccgaagctga gtataggggg 1260

cagttgcaaa ttgcgtcgct gaatcctgct ggcttcgcca gcttctacag gagctacatc 1320

aaccttcgtc tagtgtcacc attgtttcct gtgacaacat cagggcgacg tacctctcct 1380

ccaaccccgt ccagcaccaa cgcaccattc cacaacgttt tatgcaacgt ggcggcagac 1440

tagtttattc acacaaaaaa gatccatgca agtttccacg tgctgctgct tccaacgttg 1500

aagcgtttct tgtgtcgtac gtcgatcgta tttggatctt ctcagaaacg atagcggcga 1560

catgtcgttg tatctatgtc ttcagagcgg ttcgagtaga ttttttcctt catttttcgg 1620

actgaaaaaa agaggaaatg ctaccttcca tcgctggtac tatgtagttg ttggtgcaac 1680

agcccgcgca tttgcataag gtgccttgta tatatgtgga aacgttcggt tgctacgcgc 1740

gtacctcttc gtggtctgca cacggatacc ttaagtaagc tttaattttg cttcagttgg 1800

aagccagcca gtttacttcc cccgttcttg gctagctgaa tctcgacttc tagtttggtc 1860

ggaatttata taactttccg gcgtgtacac agcgtacgaa ccatgcatat aactttatat 1920

acctttctcg atttcttttg ctgcttgttt gctcgaccta gcgtctcacc ttgtgagttg 1980

tgcatgcggg aaatagctac tcagcagtgt ggtaatcgat ca 2022

<210> 17

<211> 441

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 17

gtacccagta taaccgtaca tgcacccaac caactatata gtgtaaggaa taaaacgacg 60

tcattagcac tggagggctg ccacgtcggc cggcggatct ttggctcgcg agtcgttgga 120

tgtggcacaa ttcagcagct caacgtctcc ctctacttcg caacagagtt tttgtttttt 180

atttgcgctg gtaaaggagt tttattccat atgcataggg ttacaatcaa gaggcaggag 240

atcctccaca cacggcggac ctcgaccaag ccatacagca gtagtgctct ctgtacgact 300

atacagggcc aaacgatctg ctaccctatt ctgcagacga ctaattttca tgggaataaa 360

aactctatct accataagat gacgaatctc agcaactata tgtccatagg ctgatctggt 420

taaactgtca ccagacaaag c 441

<210> 18

<211> 163

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 18

ctctcacaca tcatcgagtt aagtacccag tataaccgta catgcaccca accaactata 60

taatgtaagg aataaaacga cgtcattagc actggggggc tgccgcgtcg gcgatgtgac 120

acaattcagc agctcaacgt ctccctctac ttcgcaacag aga 163

<210> 19

<211> 618

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 19

gcaagacttt tttgacacta gtgtattgtt aaaaacgtct tatattacga gacgaaggga 60

gtacttaaca actcaatcta caaaaaatca tgagttgggc tctatagtct gcttcgccca 120

acaaccaggt cggactagtg tgaattactg tacaccaaaa aaattctgaa ataattactg 180

aacacgtcat gacgtgtcta cttccctcca aaagtatttc agatctacat tccgcctgtc 240

aattaagcac gaaatttgaa gccgattgtg ctgcccggcc ctactcacca ctacagaaat 300

gcctagctag cggtggcggc actaccgttc ggattctttc cagcttcctg aaatagccag 360

cttcccgcga tgccagccgc agccagcttc tgacgggagt agcgattcgt tcggcagcag 420

agagtagctc tagacaccgt acgaacttga aatagcccag gaaggaagcc ggcgtgaggg 480

agggagctag gaggagtgag ctctctggcg tggatacggc ggcaagctcg atggagcagg 540

tcgaccaggt gccgacggcg agcaacagag gaagttgagg cagggtcggg cgtccgggtg 600

cgctgaggat gggccatg 618

<210> 20

<211> 741

<212> DNA

<213> Elytrigia elongata (Elytrigia elongata)

<400> 20

tcttaaagac taaattgtag ccattatgtt ttaataataa agataaagct taagtgcaaa 60

atatctgatg tgctacagtg acaaacagta ggtcggtgct acagtgccca cctactctga 120

tgagctgccc tgatcagggg tagctcatca gccaactcat ggtgtgggcc ccttggtgca 180

cagtgctatt catatgaaga taaaggagaa gagaatagag cagctaagtg agttagttct 240

ggctgctgct gctctgagcc tattcctctc tcttccccac acaaaacact aaaggaaagg 300

gctactgatc aagcctgcac tggagcactg ggaaggacct ctcacagcca tcagattgag 360

ttcttggtgg tgttgagcta aagaaaggag atggcaggac ctgcaggtca aggtatgttt 420

atctcctaac caagtttgtg gctcaaactt gtgctaaatg atcctggagc aatccaacaa 480

tggtatcaga gcccaatagc atcatgtttg agccctataa gtaataaatc agacttggtc 540

tgttgcaatt aagttaaagt atcaagctgc tcttcctaaa gatcaaggtt tgatcttatt 600

tgagccctta tgagcaatag atcatgtctg atctgttata tataagttaa gtttttgatt 660

aagctcatgt tgttcttcct atttaagtca aggaagtcct cgaacaagat gatgtcgcct 720

atcttttcta ctactacttt g 741

Claims (9)

1. The blue grain gene for improving the content of the wheat anthocyanin is characterized in that the nucleotide sequence of the gene is one of the following sequences:

(a) As shown in SEQ ID NO: 1;

(b) The coded amino acid sequence is shown as SEQ ID NO: 3.

2. The blue grain gene according to claim 1, characterized in that the amino acid sequence of the blue grain gene is as set forth in SEQ ID NO:2, respectively.

3. An expression cassette comprising a blue-granulocyte gene, wherein the nucleotide sequence of the blue-granulocyte gene is selected from the group consisting of seq id no:

(a) As shown in SEQ ID NO: 1;

(b) The coded amino acid sequence is shown as SEQ ID NO: 3.

4. The expression cassette of claim 3, wherein the nucleotide sequence of the blue granule gene is as set forth in SEQ ID NO:2, respectively.

5. The expression cassette of claim 3, wherein the blue-granule gene is further operably linked to a promoter capable of driving expression thereof, wherein the promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a spatiotemporal-specific expression promoter.

6. Use of the blue grain gene of claim 1 or 2 or the expression cassette of claim 3 or 4 for increasing the anthocyanin content of wheat.

7. A method of increasing anthocyanin levels in wheat tissues or organs, the method comprising the step of co-expressing a MYB-like transcription factor and a bHLH transcription factor in tissue organs of plants, wherein the MYB-like transcription factor is a ThMYB1 gene having a nucleotide sequence selected from one of the following groups:

(a) As shown in SEQ ID NO: 1;

(b) The coded amino acid sequence is shown as SEQ ID NO: 3;

and the bHLH transcription factor is a ThR1 gene, and the nucleotide sequence of the bHLH transcription factor is one of the following sequences:

(a) As shown in SEQ ID NO: 7;

(b) The coded amino acid sequence is shown as SEQ ID NO:9, or a nucleotide sequence shown in the specification.

8. The method of claim 7, wherein the nucleotide sequence of the ThMYB1 gene is set forth in SEQ ID NO:2 and the nucleotide sequence of the ThR1 gene is shown as SEQ ID NO: shown in fig. 8.

9. Use of the method of claim 7 to increase the anthocyanin content of a wheat tissue or organ.

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